ArticlePublisher preview available

Single nucleotide polymorphism analysis for the production of valuable steroid intermediates in Mycobacterium neoaurum

Authors:
Min Liu at East China University of Science and Technology
Min Liu
Zhu Zhantao at East China University of Science and Technology
Zhu Zhantao
Xin-Yi Tao
Xin-Yi Tao
Xin-Yi Tao
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Feng-Qing Wang at East China University of Science and Technology
Feng-Qing Wang
Show all 5 authorsHide
Read publisher preview
Download citation
To read the full-text of this research, you can request a copy directly from the authors.

Abstract and Figures

Objectives To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis. Results The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism. Conclusions The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.
Figures - available from: Biotechnology Letters
This content is subject to copyright. Terms and conditions apply.
A preview of this full-text is provided by Springer Nature.
Learn more
Content available from Biotechnology Letters
This content is subject to copyright. Terms and conditions apply.
ORIGINAL RESEARCH PAPER
Single nucleotide polymorphism analysis for the production
of valuable steroid intermediates in Mycobacterium
neoaurum
Min Liu .Zhan-Tao Zhu .Xin-Yi Tao .Feng-Qing Wang .Dong-Zhi Wei
Received: 19 May 2016 / Accepted: 28 July 2016 / Published online: 29 August 2016
ÓSpringer Science+Business Media Dordrecht 2016
Abstract
Objectives To investigate single nucleotide poly-
morphism (SNP) in the transformation process of
phytosterol to valuable steroid intermediates in three
steroid-producing Mycobacterium neoaurum strains
using deep sequencing and bioinformation analysis.
Results The assembled contig sequences from RNA
sequencing of strains producing 9a-hydroxy-4-an-
drostene-3,17-dione (9OHAD), 1,4-androstadiene-
3,17-dione (ADD), and 22-hydroxy-23, 24-bisnor-
chola-1,4-dien-3-one (1,4-BNA) were analyzed for
the presence of putative SNPs for steroid catabolism.
413, 375, and 491 SNPs were detected in the coding
domain sequences and non-coding domain sequences
of RNA sequencing reads of M. neoaurum strains
producing 9OHAD, ADD, and BNA, respectively.
Special attention was focused on SNPs associated with
genes showing differential expression at proteome
level, including the genes for sterol catabolism,
glycerol catabolic process, signal transduction sys-
tems, transport system and energy metabolism.
Conclusions The work facilitates the understanding
of underlying genetic changes that may be responsible
for steroid accumulation in M. neoaurum and is useful
for its targeted genetic engineering.
Keywords Deep sequencing Mycobacterium
neoaurum RNA sequencing Single nucleotide
polymorphism Sterol transformation Steroid
Transcriptome
Introduction
Steroid pharmaceuticals are the most widely marketed
category next to antibiotics in the pharmaceutical
industry with an annual global market of about US$ 10
billion. Microbial steroid transformation is a powerful
tool for the efficient production of steroid active
pharmaceutical ingredients and key intermediates
from sterol substrates. Many steroid intermediates
with varying biochemical properties, such as 9a-
hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-an-
drostadiene-3,17-dione (ADD), and 22-hydroxy-23,
24-bisnorchola-1,4-dien-3-one (1,4-BNA), can be
obtained by the catabolism of sterols using Mycobac-
terium spp. (Wang et al. 2011; Yao et al. 2013,2014).
Advances in high-throughput sequencing have led
to the discovery of many single nucleotide
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-016-2187-z) contains supple-
mentary material, which is available to authorized users.
M. Liu Z.-T. Zhu X.-Y. Tao F.-Q. Wang (&)
D.-Z. Wei (&)
State Key Lab of Bioreactor Engineering, Newworld
Institute of Biotechnology, East China University of
Science and Technology, Shanghai 200237, China
e-mail: fqwang@ecust.edu.cn
D.-Z. Wei
e-mail: dzhwei@ecust.edu.cn
123
Biotechnol Lett (2016) 38:1881–1892
DOI 10.1007/s10529-016-2187-z
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... AD is a compound speciically used as a precursor for the majority of pharmaceutically active steroids such as testosterone, estradiol, ethinylestradiol, testolactone, progesterone, cortisone, cortisol, prednisone and prednisolone [1]. The steroid pharmaceuticals are of great importance for their role in the management of human fertility, osteoporosis, menopause and blood pressure regulation [2,3]. Commercially, steroid production represents one of the largest sectors of medical products manufactured by the pharmaceutical industry [4]. ...
... There are a number of organisms known to transform phytosterols to AD such as Aspergillus, Arthrobacter, Bacillus, Brevibacterium, Chryseobacterium, Fusarium, Gordonia, Nocardia, Pseudomonas, Rhodococcus, Streptomyces and Mycobacterium sp. [2,3]. ...
View
... Despite the fact that best practices for culturing new organisms have been developed, however, even with these best practices, the typical procedure for culturing new organism still requires a great deal of experience, and trial and error (Oberhardt, et al., 2015).Culture media, as fundamentally important as it is in microbiological tests (Cundell, 2002) requires high quality specific to the growth and preservation of microbes, the possibility of achieving accurate, reproducible and repeatable microbiological test results and other usefulness may be reduced (Sandle, 2012). Unsuitable culture media can hinder the usefulness of micro-organisms in industry (Liu et al., 2020;Mota-Gutierrez, et al., 2019;Watzlawick and Altenbuchner, 2019;Heitmann, et al., 2018), pharmaceutical (Cherukuri et al., 2020;Fernandez-Cabezon, et al., 2018;Rosenberg et al., 2017;Liu et al., 2016) and agriculture (Pham, et al., 2019) which in turn affect research, learning and economic value. Agar (agarose gel) is used for the purpose of gelling (solidifying) the microbial culture medium. ...
View
Rational development of mycobacteria cell factory for advancing the steroid biomanufacturing
Article
Full-text available
  • Aug 2022
  • WORLD J MICROB BIOT
Steroidal resource occupies a vital proportion in the pharmaceutical industry attributing to their important therapeutic effects on fertility, anti-inflammatory and antiviral activities. Currently, microbial transformation from phytosterol has become the dominant strategy of steroidal drug intermediate synthesis that bypasses the traditional chemical route. Mycobacterium sp. serve as the main industrial microbial strains that are capable of introducing selective functional modifications of steroidal intermediate, which has become an indispensable platform for steroid biomanufacturing. By reviewing the progress in past two decades, the present paper concentrates mainly on the microbial rational modification aspects that include metabolic pathway editing, key enzymes engineering, material transport pathway reinforcement, toxic metabolic intermediates removal and byproduct reconciliation. In addition, progress on omics analysis and direct genetic manipulation are summarized and classified that may help reform the industrial hosts with more efficiency. The paper provides an insightful present for steroid biomanufacturing especially on the current trends and prospects of mycobacteria.
View
New Insights on Steroid Biotechnology
Article
Full-text available
  • May 2018
Nowadays steroid manufacturing occupies a prominent place in the pharmaceutical industry with an annual global market over $10 billion. The synthesis of steroidal active pharmaceutical ingredients (APIs) such as sex hormones (estrogens, androgens, and progestogens) and corticosteroids is currently performed by a combination of microbiological and chemical processes. Several mycobacterial strains capable of naturally metabolizing sterols (e.g., cholesterol, phytosterols) are used as biocatalysts to transform phytosterols into steroidal intermediates (synthons), which are subsequently used as key precursors to produce steroidal APIs in chemical processes. These synthons can also be modified by other microbial strains capable of introducing regio- and/or stereospecific modifications (functionalization) into steroidal molecules. Most of the industrial microbial strains currently available have been improved through traditional technologies based on physicochemical mutagenesis and selection processes. Surprisingly, Synthetic Biology and Systems Biology approaches have hardly been applied for this purpose. This review attempts to highlight the most relevant research on Steroid Biotechnology carried out in last decades, focusing specially on those works based on recombinant DNA technologies, as well as outlining trends and future perspectives. In addition, the need to construct new microbial cell factories (MCF) to design more robust and bio-sustainable bioprocesses with the ultimate aim of producing steroids à la carte is discussed.
View
Two-Component Regulatory Systems of Mycobacteria
Article
Full-text available
  • Jan 2014
Two-Component Regulatory Systems of Mycobacteria, Page 1 of 2 Abstract Two-component regulatory systems (2CRSs) are widely used by bacteria to sense and respond to environmental stimuli with coordinated changes in gene expression. Systems are normally comprised of a sensory kinase protein that activates a transcriptional regulator by phosphorylation. Mycobacteria have few 2CRSs, but they are of key importance for bacterial survival and play important roles in pathogenicity. Mycobacterium tuberculosis has 12 paired two-component regulatory systems (which include a system with two regulators and one sensor, and a split sensor system), as well as four orphan regulators. Several systems are involved in virulence, and disruption of different systems leads to attenuation or hypervirulence. PhoPR plays a major role in regulating cell wall composition, and its inactivation results in sufficient attenuation of M. tuberculosis that deletion strains are live vaccine candidates. MprAB controls the stress response and is required for persistent infections. SenX3-RegX3 is required for control of aerobic respiration and phosphate uptake, and PrrAB is required for adaptation to intracellular infection. MtrAB is an essential system that controls DNA replication and cell division. The remaining systems (KdpDE, NarL, TrcRS, TcrXY, TcrA, PdtaRS, and four orphan regulators) are less well understood. The structure and binding motifs for several regulators have been characterized, revealing variations in function and operation. The sensors are less well characterized, and stimuli for many remain to be confirmed. This chapter reviews our current understanding of the role of two-component systems in mycobacteria, in particular M. tuberculosis.
View
Mycobacterium tuberculosis H37Rv has a single nucleotide polymorphism in PHoR which affects cell wall hydrophobicity and gene expression
Article
Full-text available
  • Jan 2015
Mycobacterium tuberculosis is a successful pathogen which can adapt to multiple environmental niches. As part of its repertoire of adaptive responses, two component regulatory systems play a major role in co-ordinating gene expression at the global level. The PhoPR system controls major cellular functions, including respiration, lipid metabolism, the immediate and enduring hypoxic responses, stress responses, and persistence. We identified a single nucleotide polymorphism (SNP) found in the sensor kinase (PhoR) of this system between two commonly used strains of M. tuberculosis, H37Rv (PhoRP152) and CDC1551 (PhoRL152). We constructed an isogenic strain of H37Rv carrying PhoRL152, as well as strains containing two different copies of the PhoPR locus, to determine the functional consequences of the SNP on phenotypic traits. The previously identified Apr locus was not acid-inducible in H37Rv, although it was in the CDC1551 strain. Surprisingly, the acid-responsive expression was not completely dependent on the PhoR SNP, and the locus remained constitutively expressed even in the isogenic strain H37Rv:PhoRL152. The pattern of expression in PhoPR merodiploid strains was more complex, with neither allele showing dominance. This suggests that Apr regulation is more complex than previously thought and that additional factors must be responsible for Apr up-regulation in response to acid conditions. In contrast, differences we identified in cell hydrophobicity between the two strains was wholly dependent on PhoR, confirming its role as major regulator of cell wall composition. Thus the SNP in the sensor kinase has functional consequences which account for some of the differences between widely-used laboratory strains.
View
Energy Metabolism in Mycobacterium gilvum PYR-GCK: Insights from Transcript Expression Analyses Following Two States of Induction
Article
Full-text available
Mycobacterium gilvum PYR-GCK, a pyrene degrading bacterium, has been the subject of functional studies aimed at elucidating mechanisms related to its outstanding pollutant bioremediation/biodegradation activities. Several studies have investigated energy production and conservation in Mycobacterium, however, they all focused on the pathogenic strains using their various hosts as induction sources. To gain greater insight into Mycobacterium energy metabolism, mRNA expression studies focused on respiratory functions were performed under two different conditions using the toxic pollutant pyrene as a test substrate and glucose as a control substrate. This was done using two transcriptomic techniques: global transcriptomic RNA-sequencing and quantitative Real-Time PCR. Growth in the presence of pyrene resulted in upregulated expression of genes associated with limited oxygen or anaerobiosis in M.gilvum PYR-GCK. Upregulated genes included succinate dehydrogenases, nitrite reductase and various electron donors including formate dehydrogenases, fumarate reductases and NADH dehydrogenases. Oxidative phosphorylation genes (with respiratory chain complexes I, III -V) were expressed at low levels compared to the genes coding for the second molecular complex in the bacterial respiratory chain (fumarate reductase); which is highly functional during microaerophilic or anaerobic bacterial growth. This study reveals a molecular adaptation to a hypoxic mode of respiration during aerobic pyrene degradation. This is likely the result of a cellular oxygen shortage resulting from exhaustion of the oxygenase enzymes required for these degradation activities in M.gilvum PYR-GCK.
View
Energy Metabolism in Mycobacterium gilvum PYR-GCK: Insights from Transcript Expression Analyses Following Two States of Induction.
Article
Full-text available
Mycobacterium gilvum PYR-GCK, a pyrene degrading bacterium, has been the subject of functional studies aimed at elucidating mechanisms related to its outstanding pollutant bioremediation/biodegradation activities. Several studies have investigated energy production and conservation in Mycobacterium, however, they all focused on the pathogenic strains using their various hosts as induction sources. To gain greater insight into Mycobacterium energy metabolism, mRNA expression studies focused on respiratory functions were performed under two different conditions using the toxic pollutant pyrene as a test substrate and glucose as a control substrate. This was done using two transcriptomic techniques: global transcriptomic RNA-sequencing and quantitative Real-Time PCR. Growth in the presence of pyrene resulted in upregulated expression of genes associated with limited oxygen or anaerobiosis in M.gilvum PYR-GCK. Upregulated genes included succinate dehydrogenases, nitrite reductase and various electron donors including formate dehydrogenases, fumarate reductases and NADH dehydrogenases. Oxidative phosphorylation genes (with respiratory chain complexes I, III -V) were expressed at low levels compared to the genes coding for the second molecular complex in the bacterial respiratory chain (fumarate reductase); which is highly functional during microaerophilic or anaerobic bacterial growth. This study reveals a molecular adaptation to a hypoxic mode of respiration during aerobic pyrene degradation. This is likely the result of a cellular oxygen shortage resulting from exhaustion of the oxygenase enzymes required for these degradation activities in M.gilvum PYR-GCK.
View
Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression
Article
Full-text available
  • Oct 2013
  • BMC GENOMICS
Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of [pound sign]100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB. We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression. We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent' nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype.
View
Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance
Article
Full-text available
  • Sep 2013
  • Nat Genet
The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance-associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance-associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.
View
Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols
Article
  • May 2014
  • METAB ENG
3-Ketosteroid-△(1)-dehydrogenase (KstD) is a key enzyme involved in the microbial catabolism of sterols. Here, three homologues of KstD were characterized from Mycobacterium neoaurum ATCC 25795, showing distinct substrate preferences and transcriptional responses to steroids. Single deletion of any MN-kstD failed to result in a stable and maximum accumulation of 9-OHAD due to residual KstD activities. To develop stable 9-OHAD producers, all of these MN-KstDs were inactivated, which led to about 6.02g l(-1) of 9-OHAD from 15g l(-1) of phytosterols. However, the product was mixed with 1.55g l(-1) of AD as a major by-product. To transform AD, the oxygenase component of 3-ketosteroid-9α-hydroxylase (KSH), encoded by kshA, was overexpressed. As a result, the yield of 9-OHAD increased to 7.33g l(-1) with less than 0.31g l(-1) of AD and the selectivity of 9-OHAD production was improved to 95-97% among metabolites.
View
Reliable Identification of Genomic Variants from RNA-Seq Data
Article
  • Sep 2013
  • AM J HUM GENET
Identifying genomic variation is a crucial step for unraveling the relationship between genotype and phenotype and can yield important insights into human diseases. Prevailing methods rely on cost-intensive whole-genome sequencing (WGS) or whole-exome sequencing (WES) approaches while the identification of genomic variants from often existing RNA sequencing (RNA-seq) data remains a challenge because of the intrinsic complexity in the transcriptome. Here, we present a highly accurate approach termed SNPiR to identify SNPs in RNA-seq data. We applied SNPiR to RNA-seq data of samples for which WGS and WES data are also available and achieved high specificity and sensitivity. Of the SNPs called from the RNA-seq data, >98% were also identified by WGS or WES. Over 70% of all expressed coding variants were identified from RNA-seq, and comparable numbers of exonic variants were identified in RNA-seq and WES. Despite our method's limitation in detecting variants in expressed regions only, our results demonstrate that SNPiR outperforms current state-of-the-art approaches for variant detection from RNA-seq data and offers a cost-effective and reliable alternative for SNP discovery.
View
The TetR Family of Regulators
Article
  • Sep 2013
  • MICROBIOL MOL BIOL R
SUMMARY The most common prokaryotic signal transduction mechanisms are the one-component systems in which a single polypeptide contains both a sensory domain and a DNA-binding domain. Among the >20 classes of one-component systems, the TetR family of regulators (TFRs) are widely associated with antibiotic resistance and the regulation of genes encoding small-molecule exporters. However, TFRs play a much broader role, controlling genes involved in metabolism, antibiotic production, quorum sensing, and many other aspects of prokaryotic physiology. There are several well-established model systems for understanding these important proteins, and structural studies have begun to unveil the mechanisms by which they bind DNA and recognize small-molecule ligands. The sequences for more than 200,000 TFRs are available in the public databases, and genomics studies are identifying their target genes. Three-dimensional structures have been solved for close to 200 TFRs. Comparison of these structures reveals a common overall architecture of nine conserved α helices. The most important open question concerning TFR biology is the nature and diversity of their ligands and how these relate to the biochemical processes under their control.
View
Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum
Article
  • Nov 2012
  • METAB ENG
Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87g/l androst-1,4-diene-3,17-dione and 4.53g/l androst-4-ene-3,17-dione, respectively.
View