Expression of furfural reductase improved furfural tolerance in Antarctic bacterium pseudomonas extremaustralis

Whole-cell biocatalysis using Antarctic bacteria is presently hampered by a lack of genetic information, limited gene tools and critically, a poor range of cultivation conditions. In this work, biological engineering strategy was employed for developing Pseudomonas extremaustralis, a metabolically-versatile and biopolymer-producing Antarctic bacterium, as a new whole-cell biocatalytic host. For this purpose, gene cloning and plasmid construction were carried out for overexpression of furfural reductase (FucO), an industrially-important enzyme for degradation of toxic furfural compound commonly found in lignocellulosic biorefinery. FucO gene from Escherichia coli BL21 was cloned in pJM105 plasmid and transformed into competent cells of P. extremaustralis to generate a biologically-engineered pFucO strain. For functional characterization of the enzyme, furfural reductase activity was assayed, where the P. extremaustralis pFucO strain exhibited increased furfural reductase activity of about 15.6 U/mg, an 18.8-fold higher than empty plasmid-carrying control pJM105 strain (0.83 U/mg). Furfural detoxification activity using whole cells was also determined by which the overexpression of FucO led to increased tolerance and cell growth with an OD600 value of 5.3 as compared to the control pJM105 strain that was inhibited with 10 mM furfural during 48-hour cultivation. Therefore, the findings obtained in this study successfully demonstrated the development of P. extremaustralis as biocatalytic host for the production of recombinant furfural reductase. The bioengineering would serve as a modular biotechnological platform for polar strain and bioproduct development tailored towards industrial biotechnology applications

Development of Corynebacterium glutamicum as staphylococcal-targeting chassis via the construction of autoinducing peptide (AIP)-responsive expression system

Despite increasing reports of antimicrobial activities of commensal and non-pathogenic bacteria such as Corynebacterium spp., previous studies on bioengineered therapeutics traditionally employed probiotics and food-grade bacteria which limits further advancements into microbial therapeutics research. In this study, Corynebacterium glutamicum, a generally recognised as safe (GRAS) and model bacterium was employed as a new chassis for the development of bioengineered corynebacterial chassis tailored towards Staphylococcus sp. via autoinducer peptide (AIP)-based quorum sensing (QS) interactions. To develop C. glutamicum as a staphylococcal-targeting chassis, the bacteria were transformed with the pResponse plasmid harboring AIP-responding accessory regulatory proteins agrAC and red fluorescent protein (RFP) genes under the control of the PaceA and P3 promoter, respectively, which was expected to stimulate the production of fluorescence signals in the presence of AIPs. Fluorescence activity of the C. glutamicum pResponse strain was compared to control C. glutamicum pRFP strain containing only the P3-RFP gene without the agrAC gene cassette. Using AIP-I as the input biomolecule, C. glutamicum pResponse strain fluoresced under different concentrations of AIP-I whereas no fluorescence was observed in the control C. glutamicum pRFP strain. When tested with S. aureus culture supernatant, the pResponse strain exhibited increasing fluorescence over the incubation period with the highest fluorescence signal of 183 relative fluorescence units (R.F.U) was observed at the 48 h point thereby demonstrating a functional QS-responsive protein expression system in bioengineered C. glutamicum. These findings demonstrated the feasibility and promising potential of developing bioengineered C. glutamicum as a staphylococcalresponsive and -targeting chassis

The role of Candida albicans candidalysin ECE1 gene in oral carcinogenesis

Oral squamous cell carcinoma is associated with many known risk factors including tobacco smoking, chronic alcoholism, poor oral hygiene, unhealthy dietary habits and microbial infection. Previous studies have highlighted Candida albicans host tissue infection as a risk factor in the initiation and progression of oral cancer. C. albicans invasion induces several cancerous hallmarks, such as activation of proto-oncogenes, induction of DNA damage and overexpression of inflammatory signalling pathways. However, the molecular mechanisms behind these responses remain unclear. A recently discovered fungal toxin peptide, candidalysin, has been reported as an essential molecule in epithelial damage and host recognition of C albicans infection. Candidalysin has a clear role in inflammasome activation and induction of cell damage. Several inflammatory molecules such as IL-6, IL-17, NLRP3 and GM-CSF have been linked to carcinogenesis. Candidalysin is encoded by the ECE1 gene, which has been linked to virulence factors of C albicans such as adhesion, biofilm formation and filamentation properties. This review discusses the recent epidemiological burden of oral cancer and highlights the significance of the ECE1 gene and the ECE1 protein breakdown product, candidalysin in oral malignancy. The immunological and molecular mechanisms behind oral malignancy induced by inflammation and the role of the toxic fungal peptide candidalysin in oral carcinogenesis are explored. With increasing evidence associating C albicans with oral carcinoma, identifying the possible fungal pathogenicity factors including the role of candidalysin can assist in efforts to understand the link between C albicans infection and carcinogenesis, and pave the way for research into therapeutic potentials

Current progress in production of flavonoids using systems and synthetic biology platforms

Flavonoid is an industrially-important compound due to its high pharmaceutical and cosmeceutical values. However, conventional methods in extracting and synthesizing flavonoids are costly, laborious and not sustainable due to small amount of natural flavonoids, large amounts of chemicals and space used. Biotechnological production of flavonoids represents a viable and sustainable route especially through the use of metabolic engineering strategies in microbial production hosts. In this review, we will highlight recent strategies for the improving the production of flavonoids using synthetic biology approaches in particular the innovative strategies of genetically-encoded biosensors for in vivo metabolite analysis and high-throughput screening methods using fluorescence-activated cell sorting (FACS). Implementation of transcription factor based-biosensor for microbial flavonoid production and integration of systems and synthetic biology approaches for natural product development will also be discussed

Recent advancement of engineering microbial hosts for the biotechnological production of flavonoids

Flavonoids are polyphenols that are important organic chemicals in plants. The health benefits of flavonoids that result in high commercial values make them attractive targets for large-scale production through bioengineering. Strategies such as engineering a flavonoid biosynthetic pathway in microbial hosts provide an alternative way to produce these beneficial compounds. Escherichia coli, Saccharomyces cerevisiae and Streptomyces sp. are among the expression systems used to produce recombinant products, as well as for the production of flavonoid compounds through various bioengineering approaches including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering and genetically encoded biosensors to detect flavonoid biosynthesis. In this study, we review the recent advances in engineering model microbial hosts as being the factory to produce targeted flavonoid compounds

Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae

Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study, a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S. cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAG-CAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis gene was efficiently constructed to be the starting platform for the production of plant natural products in genetically-engineered S. cerevisiae

Development of CRISPR-cas9 ribonucleoprotein complex platform for disruption of candidalysin in Candida albicans

The ECE1 gene encodes the ECE1 protein, which is processed into the toxic peptide, candidalysin in Candida albicans. Candidalysin is responsible for host epithelial cell damage and activation of the host cell inflammasome pathway during C. albicans colonization and proliferation, and is the most recent addition to the list of virulence factors in C. albicans. CRISPR-Cas9 is being increasingly used for functional studies of virulence markers especially in animals and microbes due to its simplicity and efficiency. Previous efforts using CRISPR-Cas9 for gene editing in Candida albicans has mainly used plasmid-based approaches which require serial cloning of the Cas9 and target gRNA sequences into the plasmid before transformation into competent C. albicans cells. The process is time-consuming and is associated with off- targeting, insertional mutagenesis and poor efficiency of delivery. An alternative method for plasmid-based delivery of CRISPR-Cas9 components is delivery of a Cas9-gRNA ribonucleoprotein (RNP) complex to the cells. The Cas9-gRNA RNP complex delivery method has been described for editing in non-albicans Candida, but not in C. albicans. Here we report a Cas9-gRNA RNP complex approach instead of plasmid for disruption of the ECE1 gene and insertion of a selection marker in its locus. Three gRNAs were used to target three different locations on the ECE1 gene (upstream, middle and downstream of the gene). The gRNAs were complexed with purified Cas9 protein to generate the Cas9-sgRNA RNP complex and delivered into competent C. albicans cells via electroporation. The method is a faster, simpler and more efficient alternative to plasmid- based CRISPR-Cas9 gene editing in C. albicans

Episomal and integrative DNA transformation in Candida albicans using frozen EZ Yeast transformation kit II

C. albicans is an important opportunistic fungus that is virulent in immunocompromised individuals such as HIV, cancer or transplant patients. Transformation of C. albicans is fundamental to genetic manipulation of C. albicans since it lacks a complete sexual cycle. Homologous recombination is the predominant method for transformation and expression of exogenous DNA, however, the expression of episomal plasmids have been reported. Genetic transformation of C. albicans is traditionally done via lithium acetate/spheroplast/electoporation methods that are time- consuming and/or complicated. The Frozen EZ Yeast Transformation II kit is a fast, broad spectrum, high transformation efficiency method for preparing competent cells and performing multiple plasmid transformations in yeast cells. The kit allows for easier and more efficient yeast circular and linear plasmid transformations compared to protocols. However, its effectiveness in C. albicans has not been reported. Here we report transformation of circular episomal DNA and linear integrative DNA into C. albicans using the Frozen EZ Yeast Transformation II kit. Heat shock at 44 °C, overnight incubation and outgrowth step were added as modifications specific to C. albicans transformation to increase transformation efficiency using the kit. The results with and without modification to the general kit protocol were compared. Transformation efficiency of episomal DNA using the general kit protocol was 40%, while for linear DNA, transformation did not occur. Addition of C. albicans- specific steps to the transformation protocol increased transformation efficiency. In conclusion, the Frozen EZ Yeast Transformation II kit is suitable for C. albicans transformation of circular DNA. Addition of C. albicans-specific modification steps increases transformation efficiency of C. albicans using the kit

Development of CRISPR-Cas9-mediated site-directed mutagenesis of ECE1 gene (candidalysin) in Candida albicans

CRISPR-Cas9 has been extensively used in microbial editing considering its specificity and versatility. Cumbersome editing in C. albicans due to its aberrant chromosomal ploidy and lock of meiosis can be potentially solved using CRISPR-Cas9. In this study, the third KR (lysine-arginine) motif on the ECEI gene that encodes for candidalysin in C.olbicans is targeted. The third KR motif (AAGAGA) is substituted into KA motif (AAGGCA) through transformation of CRISPR-Cas9 ribonucleoprotein (RNP) complex, ssDNA and pYM70 that confers hygromycin B resistance into competent cells of C. albicans using electroporation. The cleaving efficiency of the designed sgRNA is tested using in vitro nuclease assay against several concentrations of RNP prior transformation. The RNP complex that formed through co-incubation of sgRNA and purified Cas9 induced double-stranded break to the nearest protospacer adjacent motif to the third KR site. The DNA break is repaired by co-transformed ssDNA that contains KA motif at the third KR nucleotides flanked by 90 bp homologous arms via homologous recombination. The transformants were grown on YPD infused hygromycin B (600 µg/ml) for growth and selection. Mutation in successfully grown colonies will be verified through sequencing. The designed sgRNA shows in vitro efficient cutting at RNP concentrations of 15 nM, 20 nM and 25 nM. CRISPR-Cas9 is expected to direct correct substitution of bases that encodes for alanine at the third KR motif of the ECE1 gene. This study shall contribute to new approaches of gene editing and site-directed mutagenesis in C. albicons as well as in other in vitro and in vivo models