Engineering Clostridium Strain to Accept Unmethylated DNA

It is difficult to genetically manipulate the medically and biotechnologically important genus Clostridium due to the existence of the restriction and modification (RM) systems. We identified and engineered the RM system of a model clostridial species, C. acetobutylicum, with the aim to allow the host to accept the unmethylated DNA efficiently. A gene CAC1502 putatively encoding the type II restriction endonuclease Cac824I was identified from the genome of C. acetobutylicum DSM1731, and disrupted using the ClosTron system based on group II intron insertion. The resulting strain SMB009 lost the type II restriction endonuclease activity, and can be transformed with unmethylated DNA as efficiently as with methylated DNA. The strategy reported here makes it easy to genetically modify the clostridial species using unmethylated DNA, which will help to advance the understanding of the clostridial physiology from the molecular level

Fenians and Ribbonmen: The Development of Republican Politics in East Tyrone, 1898-1918 [Book review]

Development of sustainable processes to produce bio-based compounds is necessary due to the severe environmental problems caused by the use of fossil resources. Metabolic engineering can facilitate the development of highly efficient cell factories to produce these compounds from renewable resources. The objective of systems biology is to gain a comprehensive and quantitative understanding of living cells and can hereby enhance our ability to characterize and predict cellular behavior. Systems biology of industrial microorganisms is therefore valuable for metabolic engineering. Here we review the application of systems biology tools for the identification of metabolic engineering targets which may lead to reduced development time for efficient cell factories. Finally, we present some perspectives of systems biology for advancing metabolic engineering further

Recent Advances in Directed Yeast Genome Evolution

Saccharomyces cerevisiae, as a Generally Recognized as Safe (GRAS) fungus, has become one of the most widely used chassis cells for industrial applications and basic research. However, owing to its complex genetic background and intertwined metabolic networks, there are still many obstacles that need to be overcome in order to improve desired traits and to successfully link genotypes to phenotypes. In this context, genome editing and evolutionary technology have rapidly progressed over the last few decades to facilitate the rapid generation of tailor-made properties as well as for the precise determination of relevant gene targets that regulate physiological functions, including stress resistance, metabolic-pathway optimization and organismal adaptation. Directed genome evolution has emerged as a versatile tool to enable researchers to access desired traits and to study increasingly complicated phenomena. Here, the development of directed genome evolutions in S. cerevisiae is reviewed, with a focus on different techniques driving evolutionary engineering

An Ultrafast Hydrogel Photocrosslinking Method for Direct Laser Bioprinting

Photocrosslinking is a widely-used method to generate cell-laden hydrogels for tissue engineering. At present, it usually takes more than 30 seconds to crosslink hydrogels using UV illumination, and this delay makes it more likely that damage will occur in the DNA. With this in mind, we introduce an ultrafast photocrosslinking method using a low-cost blue laser diode. Experimental results show that a hydrogel with a diameter of 8 mm can be crosslinked using this process within 10 seconds with over 90% cell viability. Moreover, it is shown that the laser can be focused for the implementation of bioprinting. A microscale cell-laden microtube was successfully fabricated with this laser-based system, demonstrating its feasibility for bioprinting.Applied Science, Faculty ofEngineering, School of (Okanagan)ReviewedFacultyResearche

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant <it>Clostridium acetobutylicum</it> Rh8 switches ABE fermentation to high level IBE fermentation

Abstract Background Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of thl promoter. Results The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach switched the traditional ABE (acetone-butanol-ethanol) fermentation to IBE (isopropanol-butanol-ethanol) fermentation. The total alcohol titer reached 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. Conclusions The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 can thus be considered as a good host for further engineering of solvent/alcohol production.</p

Functional pyruvate formate lyase pathway expressed with two different electron donors in <i>Saccharomyces cerevisiae</i> at aerobic growth

Pyruvate formate lyase (PFL) is characterized as an enzyme functional at anaerobic conditions, since the radical in the enzyme's active form is sensitive to oxygen. In this study, PFL and its activating enzyme from Escherichia coli were expressed in a Saccharomyces cerevisiae strain lacking pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc-MTH1-Delta T ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression of either of these electron donors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenases. Among the two electron donors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin

Construction and testing of Yarrowia lipolytica recombinant protein expression chassis cells based on the high-throughput screening and secretome

Abstract Background In the recombinant protein market with broad economic value, the rapid development of synthetic biology has made it necessary to construct an efficient exocrine expression system for the different heterologous proteins. Yarrowia lipolytica possesses unique advantages in nascent protein transport and glycosylation modification, so it can serve as a potential protein expression platform. Although the Po1 series derived from W29 is often used for the expression of the various heterologous proteins, the ability of W29 to secrete proteins has not been verified and the Po1 series has been found to be not convenient for further gene editing. Results A total of 246 Y. lipolytica strains were evaluated for their secretory capacity through performing high-throughput screening in 48-well plate. Thereafter, following two rounds of shake flask re-screening, a high-secreting protein starting strain DBVPG 5851 was obtained. Subsequently, combined with the extracellular protein types and relative abundance information provided by the secretome of the starting strain, available chassis cell for heterologous protein expression were preliminarily constructed, and it was observed that the most potential signal peptide was derived from YALI0D20680g. Conclusions This study offers a novel perspective on the diversification of Y. lipolytica host cells for the heterologous protein expression and provides significant basis for expanding the selection space of signal peptide tools in the future research

Genetic manipulation of <i>C. acetobutylicum</i> SMB009 using unmethylated DNA.

<p><b>A.</b> Overexpression of <i>fdh</i> gene in <i>C. acetobutylicum</i> SMB009. The cells were grown to OD600 = 2∼3 in mRCM broth containing 50 µg/ml of erythromycin at 37°C. The boiled cell lysates were analyzed by SDS-PAGE (12% polyacrylamide gel). The overexpressed protein FDH (theoretical molecular weight 40 kDa predicted by DNAMAN Version 5) is indicated by the arrow on the right. <b>B.</b> Disruption of <i>adc</i> gene in <i>C. acetobutylicum</i> SMB009. Insertion of CTermB fragment into <i>adc</i> ORF was confirmed by PCR using the primers of 12-adc1 and 13-adc2 (corresponding to <i>adc</i> ORF positions 41-58 and 523-540 respectively). The insertion site of CTermB in <i>adc</i> ORF was validated by sequencing the PCR product of strain SMB009(<i>adc</i>::CTermB) amplified using the primers of 12-adc1 and 13-adc2.</p

Detection of Cac824I activity in <i>C. acetobutylicum</i> mutant SMB009 using methylated or unmethylated pMTL007 as DNA substrate.

<p><b>A.</b> Digestion of pMTL007 using the whole cell extracts of the wild type strain DSM1731 and the mutant SMB009 respectively. <b>B.</b> Digestion of pMTL007 using the protoplast extracts of the wild type strain DSM1731 and the mutant SMB009.</p