Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis

14α-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochlioboluslunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C.lunatus and produced in high yields 14αOH-AD and 14αOH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.

Construction of high copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980

Background: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli - B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence ( ori ) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1~pUC980-8) containing all possible insertion sites and directions were constructed.Results: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN , an alkaline protease spro1 and a pullulanase gene pulA11 , respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were upto 5200 U/mL, 21537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports.Conclusion: In this work we can conclude that the optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis . And the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

Identification of a cis-Acting Element Derived from Tomato Leaf Curl Yunnan Virus that Mediates the Replication of a Deficient Yeast Plasmid in Saccharomyces cerevisiae

Geminiviruses are a group of small single-stranded DNA viruses that replicate in the host cell nucleus. It has been reported that the viral replication initiator protein (Rep) and the conserved common region (CR) are required for rolling circle replication (RCR)-dependent geminivirus replication, but the detailed mechanisms of geminivirus replication are still obscure owing to a lack of a eukaryotic model system. In this study, we constructed a bacterial–yeast shuttle plasmid with the autonomous replication sequence (ARS) deleted, which failed to replicate in Saccharomyces cerevisiae cells and could not survive in selective media either. Tandemly repeated copies of 10 geminivirus genomic DNAs were inserted into this deficient plasmid to test whether they were able to replace the ARS to execute genomic DNA replication in yeast cells. We found that yeast cells consisting of the recombinant plasmid with 1.9 tandemly repeated copies of tomato leaf curl Yunnan virus isolate Y194 (TLCYnV-Y194, hereafter referred to as Y194) can replicate well and survive in selective plates. Furthermore, we showed that the recombinant plasmid harboring the Y194 genome with the mutation of the viral Rep or CR was still able to replicate in yeast cells, indicating the existence of a non-canonic RCR model. By a series of mutations, we mapped a short fragment of 174 nucleotides (nts) between the V1 and C3 open reading frames (ORFs), including an ARS-like element that can substitute the function of the ARS responsible for stable replication of extrachromosomal DNAs in yeast. The results of this study established a geminivirus replication system in yeast cells and revealed that Y194 consisting of an ARS-like element was able to support the replication a bacterial–yeast shuttle plasmid in yeast cells.

Transfection CXCR3 Gene into Lymphocytic Leukemia Cell Lines By Lentiviral Vector

Objective: Chemokine receptors and their ligands play important roles in the leukemia-initiating cell activity ,BCP-ALL progression and CNS infiltrating. To study the role of chemokine receptor CXCR3 in the mechanism of leukemia cell infiltration of the CNS, CXCR3 overexpression cell lines should be established. Lentivirus generally does not interfere with proto-oncogene and can express long-term and stable, so it is a good vector for genetic research on hematopoietic cells and T cells. Methods: Mouse CXCR3 gene fragment was extracted by PCR from T vector which was kept in our lab. After the extraction, CXCR3 fragment and linearized LV5 lentivirus overexpressing vector(with GFP+-puro) were digested with NotI and BamHI.After this,we used T4 DNA ligase to connect them, making the target gene (CXCR3) cloned into LV5 vector, to obtain recombinant shuttle plasmid. Next,we mixed the shuttle plasmid with packaging plasmids (pGag/Pol, pRev, pVSV-G), packaged lentivirus, collected, tested viral titer and sequenced target gene by DNA sequencing. Then the constructed CXCR3 overexpression lentivirus transfected Jurkat and L1210 cell lines, at the same time MOI achieved to 100, twice infection and adding polybrene 5ug/ml to increase lentivirus transfection efficiency. After GFP expression was observed under fluorescent microscope, we used puromycin to select transfected cells and tested the expression of CXCR3 on cell surface by flow cytometry analysis of Anti-Mouse CD183 (CXCR3). Results: After we sequenced the target mouse CXCR3 gene, viral titer reached to 1×10^9 TU/ml and GFP expression was observed after transfection 96h (<10%) on lymphocytic leukemia cell lines. Through puromycin selection, GFP+ cells surpassed 90% and CXCR3 overexpression group had upregulated 30%-90% compared with control group. Conclusion: Chemokine receptor CXCR3 had been successful transfected into lymphocytic leukemia cell lines and had stably high expression. The effect of CXCR3 in leukemic invasion and leukemia progression can be further studied using constructed CXCR3 overexpression lymphocytic cell lines. Disclosures No relevant conflicts of interest to declare.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

ABSTRACTOral pathogens, includingTreponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles ofT. denticolain microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studiedT. denticolastrain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems inT. denticolacontributed to these problems. To facilitate further molecular genetic analysis ofT. denticolabehavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation ofT. denticolaATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity inT. denticolaof a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to theTreponematoolset will enable more-rigorous and -detailed studies of the behavior of this organism.