Role Identification and Application of SigD in the Transformation of

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Role identification and application of SigD in the transformation of soybean phytosterol to 9#-hydroxy-4androstene-3,17-dione in Mycobacterium neoaurum Liang-Bin Xiong, Hao-Hao Liu, Li-Qin Xu, Dong-Zhi Wei, and Feng-Qing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05314 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Journal of Agricultural and Food Chemistry

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Role identification and application of SigD in the transformation of soybean

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phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycobacterium neoaurum

3 4

Liang-Bin Xiong,† Hao-Hao Liu,† Li-Qin Xu,† Dong-Zhi Wei,† and Feng-Qing

5

Wang*,†

6 7

†

8

Biotechnology, East China University of Science and Technology, Shanghai, China

State Key Laboratory of Bioreactor Engineering, Newworld Institute of

9 10

Corresponding Author

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* (F.Q.W.) Phone: +86 021 6425 3287. E-mail: [email protected].

12 13

Author contributions: L.B.X. and F.Q.W. designed research; L.B.X. and H.H.L.

14

performed research; L.B.X. and L.Q.X analyzed data; L.B.X., D.Z.W., and F.Q.W.

15

wrote the paper.

1

ACS Paragon Plus Environment


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ABSTRACT: 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid

17

pharmaceutical intermediate which can be produced by the conversion of soybean

18

phytosterols in mycobacteria. However, the unsatisfactory productivity and

19

conversion efficiency of engineered mycobacterial strains hinder their industrial

20

applications. Here, a sigma factor D (sigD) was investigated due to its dramatic

21

down-regulation during the conversion of phytosterols to 9-OHAD. It was determined

22

as a negative regulator in the metabolism of phytosterols and the deletion of sigD in a

23

9-OHAD-producing strain significantly enhanced the titer of 9-OHAD by 18.9%.

24

Furthermore, a high yielding strain was constructed by the combined modifications of

25

sigD and choM2, a key gene in the phytosterol metabolism pathway. After the

26

modifications, the productivity of 9-OHAD reached 0.071 g/L/h (10.27 g/L from 20

27

g/L phytosterol), which was 22.5% higher than the original productivity of 0.058

28

g/L/h (8.37 g/L from 20 g/L phytosterol) in the industrial resting cell

29

bio-transformation system.

30

mycobacteria,

9α-hydroxy-4-androstene-3,17-dione

31

KEYWORDS:

32

soybean phytosterol, transcription factor, sigD, choM2

(9-OHAD),

2


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INTRODUCTION

34

Mycobacteria can utilize natural sterols as carbon and energy sources.1,2 Inhibition of

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sterol catabolic pathway in mycobacteria leads to the accumulation of some valuable

36

steroid pharmaceutical intermediates, such as 4-androstene-3,17-dione (AD),

37

1,4-androstadiene-3,17-dione

38

(9-OHAD).3−5 These compounds produced by the biotransformation of sterols have

39

been used as commercial precursors to synthesize steroid drugs.6−8 By the way, the

40

low price soybean phytosterol is one of the preferred substrate for the industrial

41

conversion of sterols to target steroid intermediates because of its good bioavailability

42

in mycobacteria.9

(ADD),

and

9α-hydroxy-4-androstene-3,17-dione

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Since the gene cluster of sterol catabolism has been revealed in mycobacteria,10 the

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sterol catabolic pathway, especially the metabolic path from sterol substrates to

45

valuable intermediates, has been well described.11−15 Subsequently, the genetic

46

modifications of the metabolic pathway promoted the development of engineered

47

strains for the production of these high value-added metabolic intermediates of

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sterols.13−16 By the combined modifications of 3-ketosteroid-△1-dehydrogenases

49

(kstDs) and 3-ketosteroid-9α-hydroxylases (kshAs and kshBs), which are key enzymes

50

to initiate the degradation of the steroid nucleus, mycobacterial strains can be

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developed to selectively produce AD, ADD, and 9-OHAD.13−15 The disruptions of

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genes hsd4A and fadA5 involved in the C17 side chain degradation of sterols, result in

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the accumulation of a truncated side-chain, 23,24-bisnorcholenic steroids (HBCs),

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which can be used as precursors to synthesize progestogens and corticosteroids.16 3



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Although the above strategies have been successfully used to edit the sterol metabolic

56

pathway to obtain engineered mycobacterial strains with good selectivity and high

57

titer of target products, the enhanced metabolic capacity of sterols is still a continuous

58

pursuit for these engineered strains in order to further improve the productivity with

59

lower production costs.

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In general, the wild type mycobacteria digest sterols for growth without obvious

61

accumulation of intermediates.1,2 Thus, the accumulation of metabolic intermediates

62

in modified mycobacterial cells remains a serious challenge for microbial respiration

63

and growth due to the potential toxicity.17,18 Therefore, we hypothesized that the

64

engineered mycobacterial strains evolved sophisticated regulation mechanisms to

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adapt themselves to the massive accumulation of toxic metabolites in cells.

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Meanwhile, these mechanisms might inhibit the sterol catabolic pathway, thus

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affecting the productivity of target intermediates. We had confirmed that the complex

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noncoding small RNAs were involved in the metabolism regulation of phytosterols in

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mycobacteria.19 However, the metabolic regulation mechanism of phytosterols in

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mycobacterial cells was not thoroughly explored, thus hampering the efforts to further

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enhance the productivity of phytosterol metabolism.

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Regulatory factors are generally associated with key metabolic pathways. It had

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been verified that some sigma factors played key roles in the metabolism regulation of

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carbon sources.20,21 Modifications of these transcriptional regulators significantly

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changed the carbon metabolism efficiency. Knockout of sigD or sigW gene could alter

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the carbon flux distribution and significantly improve the metabolic efficiency in B. 4


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subtilis.20 Overexpression of sigE widely changed sugar metabolism and resulted in a

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2.3-fold enhancement of polyhydroxyalkanoate (PHA) production in Synechocystis sp.

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PCC 6803.21 So far, sigma factors involved in the regulation of sterol catabolism in

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mycobacteria have not been reported. In this study, we attempted to search for similar

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functional sigma factors involved in the regulation of sterol metabolism. In addition,

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such sigma factors would be potential targets to enhance the metabolic efficiency

83

from phytosterols to high value-added products.

84

Here, a 9-OHAD-producing strain, M. neoaurum ∆kstD1, was used as a model to

85

screen sigma factors related to phytosterol catabolism by RNA-sequencing analysis.

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The dramatically down-regulated gene sigD was selected to be analyzed and its

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important regulatory function was found in the transcription of some key genes

88

involved in the metabolic pathway of phytosterol to 9-OHAD. Accordingly, an

89

effective strategy to enhance the transformation of phytosterol to the high value-added

90

product, 9-OHAD, was developed by the modification of sigD, which might not be

91

directly implicated in the substrate uptake or metabolism. Moreover, some key

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enzymes involved in the catabolism of sterol to 9-OHAD were tested to further

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enhance the productivity of the 9-OHAD-producing strain.

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MATERIALS AND METHODS

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Strains, Plasmids, and Reagents. All modified strains and plasmids used in this

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work are listed in Table S1. Escherichia coli DH5α was used for plasmid replication.

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All reagents and substrates were prepared as previously described.14,15

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Bacterial Culture. Escherichia coli DH5α was cultured in Luria-Bertani (LB) 5



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medium (10.0 g/L tryptone, 5.0 g/L yeast extract, 10 g/L NaCl, pH 7.0). All

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mycobacterial strains were first recovered in LB medium. Medium MYC/01 (20.0 g/L

101

glycerol, 2.0 g/L citric acid, 0.05 g/L ammonium ferric citrate, 0.5 g/L K2HPO4, 0.5

102

g/L MgSO4·7H2O, 2.0 g/L NH4NO3, pH 7.5) was used to cultivate the mycobacterial

103

strains. The cultivated strains were then transferred into MYC/02 medium (10.0 g/L

104

glucose, 2.0 g/L citric acid, 0.05 g/L ferric ammonium citrate, 0.5 g/L MgSO4·7H2O,

105

2.0 g/L NH4NO3, pH 7.5) with 2 g/L of phytosterols for shake-flask cultivation.

106

In-frame Deletion of Target Genes. To construct in-frame deletion mutants of

107

targeted genes, the unmarked homologous recombination strategy proposed by

108

Gordhan and Parish22 was adopted in this study. To delete sigD, a 996-bp upstream

109

sequence and a 1032-bp downstream sequence were selected as recombination

110

fragments. The two fragments were cloned with the designed primer pairs

111

(D-sigD-UF & D-sigD-UR, D-sigD-DF & D-sigD-DR) in Table S2. Then the two

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fragments were dissected with the designed restriction enzymes and ligated into the

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plasmid p2NIL. Subsequently, a selection marker cassette from pGoal19 was digested

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with Pac I and then inserted into the Pac I site of p2NIL, which carried the 2028-bp

115

fragment, to construct the homologous recombination plasmid. The constructed

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plasmid was transferred into mycobacterial cells via electroporation and the

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sigD-disrupted strains were selected according to the previously reported protocol.14,15

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Complementation of Target Genes in M. neoaurum. To complement the effect of

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target genes, the expression cassette of the target gene was reconstructed and

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complemented in the deletion strain according to the previously reported method.15 6


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The complete sequence of sigD was amplified from M. neoaurum ATCC 25795 with

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the primer pairs (O-sigD-F and O-sigD-R) in Table S2. After digestion with EcoR I

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and Sal I, the fragment was inserted into the corresponding site of pMV261 to create

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an expression plasmid, p261-sigD. Subsequently, the expression cassette of sigD

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containing a heat shock promoter hsp60 from p261-sigD was digested with Xba I and

126

Sal I and then integrated into the corresponding site of pMV306 to obtain a plasmid of

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p306-sigD. Then, the p306-sigD was transferred into the sigD-deleted strain to

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complement the function of SigD.

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RNA Extraction, RNA Sequencing, and qRT-PCR Analysis. Total RNA was

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extracted with FastRNATM Pro Blue Kit for microbes and FastPrep-24TM Instrument

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(MP Biomedicals, CA) according to the manufacturer’s instructions. NanoDrop

132

(NanoDrop Technologies, MA) was employed to verify the concentration of each

133

RNA sample. FastQuant cDNA kit (TianGen Co., Ltd., Beijing, China) was used to

134

exclude the contamination of genomic DNA. Then, 1 µg total RNA and 2 µL of

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gDNA buffer was diluted to the volume of 10 µL with RNase-free ddH2O. Then, the

136

reaction mixture was incubated at 42 °C for 3 min.

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For RNA sequencing, total RNA obtained after removing DNA was taken for

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preparing the RNA-sequencing library according to the manufacturer’s instruction

139

(www.illumina.com). RNA sequencing was conducted with an Illumina HisSeqTM

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2000 (Illumina Inc., CA) according to the manufacturers’ protocols.

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For qRT-PCR analysis, 2 µL of 10×Fast RT buffer, 1 µL of RT Enzyme Mix, 2 µL

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of FQ-RT Primer Mix and 15 µL of RNase-free ddH2O was added into total RNA 7



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obtained after removing DNA. This reverse transcription reaction mixture was

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incubated to prepare the cDNA according to the following procedure: 42 °C for 15

145

min and 95 °C for 3 min. Real-time qPCR analyses of cDNA samples were performed

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on the StepOneTM Real Time PCR System (Applied Biosystems, CA). The qRT-PCR

147

reaction mixture was prepared with 10 µL of Super Real Premix Plus with SYBR

148

Green I, 0.3 µM forward primer and reverse primer, 1.6 µL of ROX Reference Dye,

149

and 50 ng cDNA, and then diluted to 20 µL with RNase-free ddH2O. The

150

amplification was performed according to the following procedure: 10 min

151

pre-denaturation, 40 cycles of denaturation at 95 °C for 15 s, annealing at 56 °C for 15

152

s, and extension at 72 °C for 30 s. Melting curve analyses were performed at the end

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of each cycle. Expression of 16S rRNA was used as the internal standard for

154

normalizing the qRT-PCR data. Relative gene expression was calculated with the

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2-∆∆CT algorithm.23 Gene expression data were determined with three independent

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replicates for each strain.

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Sterol Transformation, Product Extraction, and Analysis. Transformation

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capability of engineered strains was identified in MYC/02 medium with 2 g/L

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phytosterols as previously described.15 The industrial application potential was

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assessed in the HP-β-CD-resting cell reaction system with 20 g/L of phytosterols as

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previously described.24 The conversion progress was sampled every 24 h and the

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samples were extracted with the same volume of ethyl acetate. The extracts were

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transferred into clean tubes, dried under vacuum, re-dissolved in methanol, and

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centrifuged at 12000×g for 20 min. 8


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In high performance liquid chromatography (HPLC) analysis, the prepared samples

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containing the 9-OHAD were analyzed by using a reversed-phase C18-column (250

167

nm × 4.6 nm) (Agilent Technologies, CA) at 254 nm with an Agilent 1100 series

168

HPLC (Agilent Technologies, CA). A mixture of methanol and water (80:20, v/v) was

169

used as the mobile phase.

170

For gas chromatography (GC) analysis, a GC system 7820A (Agilent Technologies,

171

CA) was used for the quantitative determination of the mixture of phytosterols as

172

described previously.14 The ethyl acetate extracts (5 µL) of the samples were injected

173

into DB-5 column (30 m × 0.25 µm (i.d.) × 0.25 µm film thickness, Agilent

174

Technologies, CA). An initial oven temperature of 200 °C was maintained for 2 min,

175

then increased to 280 °C at the rate of 20 °C/min, maintained for 2 min, increased to

176

305 °C at 20 °C/min and maintained for 10 min. Inlet and flame-ionization detector

177

temperatures were maintained at 320 °C. Nitrogen carrier gas flow was 2 mL/min at

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50 °C.

179

RESULTS AND DISCUSSION

180

Screening of Sigma Factors Implicated in Sterol Metabolism. In the genome of

181

M. neoaurum ATCC 25795, 29 genes were predicted to encode sigma factors-related

182

proteins (Table S3). To evaluate the role of sigma factors in the metabolic pathway of

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phytosterols, the transcriptional levels of these genes in M. neoaurum ATCC 25795

184

(the wild type strain, WT) and its derivative 9-OHAD-producing strain ∆kstD1 were

185

compared in the presence or absence of phytosterol substrate (Figure 1).

186

Most of the 29 genes in WT showed transcriptional differences in the 9



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presence/absence of phytosterols (Figure 1A), and we focused on the genes whose

188

transcriptional levels significantly fluctuated after the complete degradation pathway

189

of sterol was blocked and led to mass accumulation of the metabolic intermediate

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9-OHAD in strain ∆kstD1 (Figure 1B). Among these genes, sigD (Mn_4463) is an

191

interesting gene. Its transcription only displayed slight up-regulation (1.2-fold) in WT

192

in the presence of phytosterols compared to that in the absence of phytosterols (Figure

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1A). In contrast, the gene was down-regulated by 7.7-fold in the ∆kstD1 mutant strain

194

compared with the WT in the presence of phytosterols (Figure 1B). As the deletion of

195

kstD1 blocked the complete degradation pathway of sterol and led to mass

196

accumulation of the metabolic intermediate 9-OHAD, the remarkable transcription

197

fluctuation of this gene might be ascribed to an adaptive mechanism in response to the

198

stress from the production of 9-OHAD.

199

SigD is a ubiquitous gene encoding a sigma factor D in microorganisms. It has been

200

identified as an important conserved gene in regulating global flux partitioning

201

between catabolism and anabolism in B. subtilis.20 The sigD from M. neoaurum

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ATCC 25795 (GeneBank: NZ_JMDW01000008.1; Region: 548346…548846) is

203

501-bp long and shares high sequence identity with its homologs from M.

204

tuberculosis H37Rv (639-bp, 74%) and M. smegmatis str. mc2 155 (579-bp, 81%),

205

though it is significantly shorter than the two homologs (Figure 2). Moreover, the

206

genomic location of sigD is highly conserved among mycobacteria and the sigD holds

207

similar organization frame with its flanking genes between guaB and whiB in the

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genome among different mycobacteria. The high conservation implicates its important 10


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role in cell metabolism in mycobacteria. Meanwhile, the transcriptional changes

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indicated that sigD might be an important factor involved in the regulation of

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phytosterol metabolism to produce 9-OHAD in the 9-OHAD-producing strain.

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Effect of SigD on the Conversion of Sterol to 9-OHAD. To clearly evaluate the

213

role of SigD in the conversion of phytosterols to 9-OHAD, the previously constructed

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strain ∆kstD1&2&3 was selected as a chassis because the further degradation of

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9-OHAD was almost completely blocked by inactivating all the identified kstDs in

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this strain.15 According to the targeted gene knockout method, sigD was in-frame

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deleted from the genome of the strain ∆kstD1&2&3, thus resulting in the strain

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∆kstD1&2&3&sigD (Figure 3A). In contrast, the growth rate of ∆kstD1&2&3&sigD

219

showed a slight decline compared with that of the strain ∆kstD1&2&3 (Figure 3B).

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Meanwhile, the phytosterol uptake rate did not show an obvious enhancement (Figure

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3C). However, the transformation efficiency from phytosterols to 9-OHAD in the

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strain ∆kstD1&2&3&sigD was significantly higher than that in the strain

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∆kstD1&2&3 and showed the titer improvements of 9-OHAD: 110% at 24 h, 43% at

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48 h, and 20% at 72 h, respectively (Figure 3D).

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To verify the effect of SigD on the conversion of phytosterols to 9-OHAD, the sigD

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gene was then complemented into ∆kstD1&2&3&sigD under the control of a heat

227

shock promoter hsp60 to generate a strain ∆kstD1&2&3&sigD+sigD. Comparison

228

results showed that the phenotype of the strain ∆kstD1&2&3&sigD+sigD was

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restored to that of the strain ∆kstD1&2&3 (Figures 3B, 3C and 3D). These data

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confirmed that SigD did affect the conversion of phytosterols to 9-OHAD. The 11



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dramatic enhancement of 9-OHAD titer by the deletion of sigD indicated that the

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SigD played a negative regulation role in the conversion of phytosterols to 9-OHAD.

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Effect of SigD Disruption on the Transcription Levels of Key Genes Involved

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in Sterol Metabolism. To further determine the effect of deleting sigD on phytosterol

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metabolism of the engineered strain, the transcriptional levels of some key genes

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involved in the conversion process of phytosterols to 9-OHAD were investigated in

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the strain ∆kstD1&2&3 and the strain ∆kstD1&2&3&sigD. These genes included the

238

initiation genes of sterol catabolism (cholesterol oxidase genes choM1 and choM2),14

239

the key oxidation gene of steroid nucleus (kshA), and the gene of C17 side-chain

240

degradation (17β-hydroxysteroid dehydrogenase, hsd4A) (Figure 4A).16

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ChoM1 and ChoM2 have been identified as two important enzymes synergistically

242

catalyzing the initial oxidation of sterol into sterone, which is a rate-limiting step in

243

the catabolic pathway of sterol in M. neoaurum ATCC 25795.14 Although the deletion

244

of sigD did not increase the transcription of choM2, the transcription of choM1 was

245

significantly enhanced (Figure 4B). The choM1 was up-regulated 2.2-fold and

246

3.5-fold in the strains ∆kstD1&2&3 and ∆kstD1&2&3&sigD, respectively, compared

247

with that of WT in the presence of phytosterols. In other words, the deletion of sigD

248

in the strain ∆kstD1&2&3 up-regulated the transcriptional level of choM1 (1.6-fold).

249

The transcription change of choM1 in the kstD1&2&3 deleted strain might be a kind

250

of adaptation remodeling mechanism and the further transcriptional up-regulation of

251

choM1 resulting from the deletion of sigD indicated an increased metabolic efficiency

252

of phytosterols to 9-OHAD by enhancing the rate-limiting conversion step from sterol 12


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to sterone.

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KshA is a critical enzyme which is in charge of the 9α-hydroxylation of

255

3-ketosteroids in the catabolic process of sterols. Coordinated with KstDs, KSH plays

256

an essential role in the cleavage of the steroid nucleus.18 Therefore, a

257

9-OHAD-producing strain can be obtained by the deletion of kstDs (Figure 4A).15 In

258

this study, although the transcriptional level of kshA fluctuated after the deletion of

259

sigD, the slight up-regulation or down-regulation (±0.2-fold) indicated that the

260

essential 9α-hydroxylation activity of 3-ketosteroids was basically maintained at a

261

relatively constant level (Figure 4B). Moreover, Hsd4A is a key enzyme involved in

262

the second round β-oxidation process of the sterol side chain.16 Therefore, the hsd4A

263

was selected to assess the effect of SigD on the side chain degradation of sterol. The

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transcriptional level of hsd4A was significantly up-regulated (2.6-fold) after the

265

deletion of sigD (Figure 4B).

266

Although the effects of deleting sigD on the transcription of these key genes

267

involved in the conversion of phytosterols to 9-OHAD were different among these

268

genes, the deletion of sigD did enhance the productivity of phytosterols to 9-OHAD

269

significantly. Regarding the key roles of ChoM1 and Hsd4A in the conversion of

270

phytosterols to 9-OHAD, the enhanced yield of 9-OHAD from the conversion of

271

phytosterols might be partially ascribed to the increased transcriptional levels of these

272

two genes.

273

Improving Productivity of 9-OHAD-Producing Strain by the Modifications of

274

sigD and choM2. In a previous study, augmentation of cholesterol oxidases (ChoM1 13



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and ChoM2) significantly enhanced the conversion of phytosterols to AD and ADD.14

276

Here, the four key enzymes mentioned in the previous section were individually

277

overexpressed to further increase the productivity of 9-OHAD in the strain

278

∆kstD1&2&3&sigD (Figure 5A).

279

The strain ∆kstD1&2&3&sigD overexpressing the four enzymes showed different

280

effects on the productivity of 9-OHAD. In the regular aqueous system emulsified with

281

Tween-80, overexpression of ChoM2 in the ∆kstD1&2&3&sigD mutant strain

282

showed the greatest enhancement (10.9%) of 9-OHAD after 144-h biotransformation,

283

followed by the overexpression of KshA (5.7%) and Hsd4A (2.3%). The

284

overexpression of ChoM1 did not show a significant enhancement on the yield of

285

9-OHAD (Figure 5B).

286

Next, the productivity of phytosterols to 9-OHAD in the modified strains was

287

evaluated with a “cyclodextrin-resting cell” system which had been employed in the

288

industry.24 We previously described the basic strategy to develop 9-OHAD-producing

289

strains by the combined modification of kstDs, which resulted in the stable

290

9-OHAD-producing strain ∆kstD1&2&3 with a titer of 6.78-7.33 g/L, a productivity

291

of 0.047-0.051 g/L/h and a molar yield of 50-55% from 15 g/L phytosterols in 144

292

h.15 Here, the yield of 9-OHAD in the strain ∆kstD1&2&3&sigD was significantly

293

higher (16.3%) than that in its parental strain ∆kstD1&2&3 during the transformation

294

of 20 g/L phytosterols within 144 h, and overexpression of ChoM2 in strain

295

∆kstD1&2&3&sigD led to a further increase (5.5% at 144 h) (Figure 5C). The highest

296

increase (57.8%) in the yield of 9-OHAD was observed in the strain 14


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∆kstD1&2&3&sigD++choM2

(overexpression

of

ChoM2

in

the

strain

298

∆kstD1&2&3&sigD) after 24-h conversion and the yield of 9-OHAD was increased

299

from 0.91 g/L to 1.44 g/L. Ultimately, the strain ∆kstD1&2&3&sigD++choM2

300

yielded 10.27 g/L 9-OHAD (95%) with a molar yield of 65.5% and a productivity of

301

0.071 g/L/h after 144-h transformation, whereas the strain ∆kstD1&2&3 yielded 8.37

302

g/L 9-OHAD with a molar yield of 53.3% and a productivity of 0.058 g/L/h. The

303

modification of sigD and choM2 markedly increased the 9-OHAD productivity of the

304

engineered strain by 22.5%.

305

These results improved the understanding of the metabolic mechanism of

306

mycobacteria for sterol and promoted the development of industrial strains for

307

producing valuable steroid pharmaceutical intermediates from the transformation of

308

sterol. As a kind of basic regulator, sigma factors play a basic role in controlling the

309

metabolic processes required for life. The number of sigma factors in microorganisms

310

correlates generally with the variability of the environment. M. neoaurum ATCC

311

25795 has a repertoire of 29 sigma factors, which are significantly more than 13

312

sigma factors in M. tuberculosis,10 demonstrating a more complex metabolic

313

regulatory mechanism to adapt to environmental changes. The maximum

314

up-regulation of sigF and sigH (Figure 1A) indicated that the utilization of sterol

315

might increase the stress on mycobacterial cells although it was used as a kind of

316

carbon source because sigF and sigH were two important regulators in response to a

317

variety of stress conditions, such as antibiotic stress and oxidative stress.25

318

As the intermediates of sterol metabolism were reported to inhibit cell growth and 15



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319

respiration, the remarkable down-regulation of sigD might be attributed to an adaptive

320

mechanism in response to the stress from the massive yield of 9-OHAD, which

321

inhibited cellular respiration and further led to a possible hypoxia stress.6,18,26 In

322

addition, some puzzles remained to be further explored. Does SigD directly or

323

indirectly involve the expression regulation of the key genes in the conversion

324

pathway of phytosterols to 9-OHAD? Is it a response of sigD to the accumulation of

325

9-OHAD? Nevertheless, it is definite that the deletion of sigD can be used as an

326

effective strategy to develop high 9-OHAD-producing strains for industrial

327

applications.

328 329

ABBREVIATIONS USED

330

9-OHAD, 9α-hydroxy-4-androstene-3,17-dione; AD, 4-androstene-3,17-dione; ADD,

331

1,4-androstadiene-3,17-dione; 9-OHADD, 9α-hydroxy-4- androstadiene-3,17-dione;

332

HBCs, 23,24-bisnorcholenic steroids; KstD, 3-ketosteroid-△1-dehydrogenase; KSH,

333

3-ketosteroid-9α-hydroxylase; PHA, polyhydroxyalkanoates; LB, Luria−Bertani;

334

SigD, sigma factor D; ChoM, cholesterol oxidase; Hsd4A, 17β-hydroxysteroid

335

dehydrogenase; HPLC, high performance liquid chromatography; GC, gas

336

chromatography.

337 338

ACKNOWLEDGEMENT

339

The authors sincerely thank T. Parish (Department of Infectious and Tropical Diseases,

340

United Kingdom) for providing the plasmids, p2NIL, and pGOAL19. 16


Page 17 of 29


341 342

ASSOCIATED CONTENT

343

Supporting Information

344

Table S1: Strains and plasmids used in this study.

345

Table S2: Primers used in this study.

346

Table S3: Identification and annotation of sigma factors−related genes.

347

Funding

348

This research was financially supported by the National Natural Science Foundation

349

of China (No. 31370080) and the National Special Fund for State Key Laboratory of

350

Bioreactor Engineering.

351

Notes

352

The authors declare no conflict of interest.

17



353

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FIGURE CAPTIONS Figure 1. Transcriptional changes of sigma factor related genes. (A) Transcript profiles of strain ∆kstD1 compared with M. neoaurum ATCC 25795 (wild type strain, WT) in the presence and absence of phytosterol. (B) Comparison of gene transcription levels of all identified sigma factors in strain ∆kstD1 versus the WT in the presence of phytosterol. All data showed here represent log2 fold change ratio of the genes expression. WT, the wild type strain was cultivated in MYC/02 medium without phytosterol addition. WT+C, the wild type strain was cultured in MYC/02 medium with phytosterol addition. ∆kstD1+C, the strain ∆kstD1 was cultured in MYC/02 medium with phytosterol addition. Data were from two independent analyses.

Figure 2. Localization of sigD gene in the genome of M. neoaurum ATCC 25795 and other mycobacteria. Orthologous counterparts were colored with grey background. The size and direction of genes were described as arrows according to the predicted genome information. The percentages, including 74% and 81%, indicate the amino acid sequence identity of SigD in M. neoaurum ATCC 25795 with homologs from other mycobacteria. GuaB2, inosine monophosphate dehydrogenase. WhiB3, redox-responsive transcriptional regulator.

Figure 3. Inactivation of sigD gene in M. neoaurum. (A) Evidence for deletion of sigD. The size of origin fragment containing sigD in wild type strain (WT) is 22


Page 23 of 29


2,385-bp and 357-bp core region of sigD was removed in frame. (B) Growth curve of the sigD-deleted and complemented strain with 0.5 g/L phytosterols addition. (C) Real-time quantitative analysis of the residual phytosterol. (D) Titer curves of 9-OHAD in the transformation of 2 g/L phytosterols in the Tween 80 emulsified aqueous system. Data are the mean standard deviations of three independent experiments.

Figure 4. Analysis of gene transcription by qRT-PCR. (A) Schematic profile of the metabolic block of sterol to accumulate intermediate 9-OHAD by deletion of kstD1, kstD2, and kstD3. (B) Transcriptional analysis of selected genes related to sterol catabolism in mycobacterial species. I, the wild type strain. II, ∆kstD1&2&3. III, ∆kstD1&2&3&sigD. Data represent the mean ± standard deviation of three measurements.

Figure 5. Productivity of 9-OHAD increased by overexpression of key genes involved

in

sterol

metabolism

pathway.

(A)

Cells

∆kstD1&2&3&sigD

overexpressing genes grew in MYC/02 medium with 2.0 g/L phytosterols for biotransformation. (B) 9-OHAD levels in the medium were detected after 48 h and 144 h of cultivation. (C) Assessment of different 9-OHAD-producing strains in a “cyclodextrin-resting cell” system with 20 g/L of phytosterol. Data represent the mean ± standard deviation of three measurements.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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TABLE OF CONTENTS

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Role Identification and Application of SigD in the Transformation of

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