Yankun Yang

A new Cry toxin with a unique two-component dependency from Bacillus sphaericus

Highly pathogenic strains of Bacillus sphaericus produce the mosquitocidal Bin proteins, but resistance to this toxin can be produced under laboratory and field conditions. Analysis of strains able to overcome this resistance revealed the presence of a previously undescribed type of two‐component toxin. One subunit, Cry48Aa1, is related to the 3‐domain crystal toxins of Bacillus thuringiensis. Uniquely for this type of protein, insect toxicity is only achieved in the presence of a second, accessory protein, Cry49Aa1. This protein is itself related to both the binary toxin of B. sphaericus and to Cry35 and Cry36 of B. thuringiensis, none of which require interaction with Cry48Aa1‐like proteins for their activity. The necessity for both Cry48Aa1 and Cry49Aa1 components for pathogenicity, therefore, indicates an unprecedented interaction to generate toxicity. Despite high potency for purified Cry48Aa1/Cry49Aa1 proteins (LC50 for third instar Culex quinquefasciatus larvae: 15.9 ng/ml and 6.3 ng/ml respectively), bacteria producing them show suboptimal mosquitocidal activity due to low‐level Cry48Aa1 production. This new toxin combination may indicate a fortuitous combination of members of the gene families that encode 3‐domain Cry toxins and Binary‐like toxins, permitting the “mix‐and‐match” evolution of a new component in the mosquitocidal armoury.— Jones G. W., Nielsen‐Leroux, C., Yang, Y., Yuan, Z., Dumas, V. F., Monnerat, R. G., Colin Berry C. A new Cry toxin with a unique two‐component dependency from Bacillus sphaericus. FASEB J. 21, 4112–4120 (2007)

The development and application of high throughput cultivation technology in bioprocess development

This review focuses on recent progress in the technology of high throughput (HTP) cultivation and its increasing application in quality by design (QbD) -driven bioprocess development. Several practical HTP strategies aimed at shortening process development (PD) timelines from DNA to large scale processes involving commercially available HTP technology platforms, including microtiter plate (MTP) culture, micro-scale bioreactors, and in parallel fermentation systems, etc., are critically reviewed in detail. This discussion focuses upon the relative strengths and weaknesses or limitations of each of these platforms in this context. Emerging prototypes of micro-bioreactors reported recently, such as milliliter (mL) scale stirred tank bioreactors, and microfludics integrated micro-scale bioreactors, and their potential for practical application in QbD-driven HTP process development are also critically appraised. The overall aim of such technology is to rapidly gain process insights, and since the analytical technology deployed in HTP systems is critically important to the achievement of this aim, this rapidly developing area is discussed. Finally, general future trends are critically reviewed.

Expression of recombinant protein using Corynebacterium Glutamicum: progress, challenges and applications

Abstract Corynebacterium glutamicum (C. glutamicum) is a highly promising alternative prokaryotic host for recombinant protein expression, as it possesses several significant advantages over Escherichia coli (E. coli), the currently leading bacterial protein expression system. During the past decades, several experimental techniques and vector components for genetic manipulation of C. glutamicum have been developed and validated, including strong promoters for tightly regulating target gene expression, various types of plasmid vectors, protein secretion systems and methods of genetically modifying the host strain genome to improve protein production potential. This review critically discusses current progress in establishing C. glutamicum as a host for recombinant protein expression, and examines, in depth, some successful case studies of actual application of this expression system. The established “expression tool box” for developing novel constructs based on C. glutamicum as a host are also evaluated. Finally, the existing issues and solutions in process development with C. glutamicum as a host are specifically addressed.

Proteolytic Stability of Insecticidal Toxins Expressed in Recombinant Bacilli

ABSTRACT The production of the vegetative mosquitocidal toxin Mtx1 from Bacillus sphaericus was redirected to the sporulation phase by replacement of its weak, native promoter with the strong sporulation promoter of the bin genes. Recombinant bacilli developed toxicity during early sporulation, but this declined rapidly in later stages, indicating the proteolytic instability of the toxin. Inhibition studies indicated the action of a serine proteinase, and similar degradation was also seen with the purified B. sphaericus enzyme sphericase. Following the identification of the initial cleavage site involved in this degradation, mutant Mtx1 proteins were expressed in an attempt to overcome destructive cleavage while remaining capable of proteolytic activation. However, the apparently broad specificity of sphericase seems to make this impossible. The stability of a further vegetative toxin, Mtx2, was also found to be low when it was exposed to sphericase or conditioned medium. Random mutation of the receptor binding loops of the Bacillus thuringiensis Cry1Aa toxin did, in contrast, allow production of significant levels of spore-associated protein in the form of parasporal crystals. The exploitation of vegetative toxins may, therefore, be greatly limited by their susceptibility to proteinases produced by the host bacteria, whereas the sequestration of sporulation-associated toxins into crystals may make them more amenable to use in strain improvement.

Cytolytic Toxin Cyt1Aa of Bacillus thuringiensis Synergizes the Mosquitocidal Toxin Mtx1 of Bacillus sphaericus

Using the shuttle vector pBU4, the mosquitocidal toxin gene mtx1 from Bacillus sphaericus strain SSII-1 was introduced into an acrystalliferous strain of B. thuringiensis both individually and in combination with the accessory protein gene p20 and the cytolytic protein gene cyt1Aa from B. thuringiensis subsp. israelensis. Bioassay results indicated that the recombinants B-pMT4(Mtx1) and B-pMT9(Mtx1), both individually containing mtx1, had moderate toxicities to binary toxin susceptible and binary toxin resistant Culex quinquefasciatus larvae during the vegetative growth stage, but that their toxicities declined rapidly during the sporulation phase. The LC50 values were 2.5 and 4.8 mg/ml respectively, against 3–4 instar susceptible and resistant larvae for the final sporulated cultures of recombinants B-pMT9(Mtx1), and little toxicity was detected for B-pMT4(Mtx1). Meanwhile, the recombinant B-pMPX2(Mtx1+Cyt1Aa) expressing Mtx1, P20 alone, and Cyt1Aa in combination had stable toxicities during both the vegetative phase and the sporulation phase, with a LC50 ranging from 0.45–0.58 mg/ml. Furthermore, expression of Cyt1Aa appeared to enhance the activity of Mtx1 to target mosquito larvae, suggesting a synergism between Cyt1Aa and Mtx1 toxins.

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system

BackgroundCorynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes.ResultsIn this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions.ConclusionsIn this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability.

Construction of genetic parts from the Corynebacterium glutamicum genome with high expression activities

ObjectiveTo construct effective genetic expression parts controlling transcription and translation initiation for synthetic biology and heterologous expression in Corynebacterium glutamicum.ResultTwelve highly expressed genes were identified from the proteomic data of C. glutamicum. Their related sequences were used to construct bicistronic genetic expression parts. Each part contain promoter, 5′-UTR, N-terminal sequence of the source gene and a conserved SD sequence, associated with target gene, forming the bicistronic expression cassette. The enhanced green fluorescent protein (EGFP) expression levels controlled by these novel parts have 1.4 to 790-fold increase in C. glutamicum compared with corresponding promoter-5′-UTR part. One of the bicistronic parts is 1.35 times the EGFP expression of the constitutive-expression pXMJ19. These bicistronic parts had expression advantage compared with conventional promoter-5′-UTR parts.ConclusionVarious genetic parts for efficient gene expression can be quickly obtained via this new method.

Transcriptome and Multivariable Data Analysis of Corynebacterium glutamicum under Different Dissolved Oxygen Conditions in Bioreactors

Dissolved oxygen (DO) is an important factor in the fermentation process of Corynebacterium glutamicum, which is a widely used aerobic microbe in bio-industry. Herein, we described RNA-seq for C. glutamicum under different DO levels (50%, 30% and 0%) in 5 L bioreactors. Multivariate data analysis (MVDA) models were used to analyze the RNA-seq and metabolism data to investigate the global effect of DO on the transcriptional distinction of the substance and energy metabolism of C. glutamicum. The results showed that there were 39 and 236 differentially expressed genes (DEGs) under the 50% and 0% DO conditions, respectively, compared to the 30% DO condition. Key genes and pathways affected by DO were analyzed, and the result of the MVDA and RNA-seq revealed that different DO levels in the fermenter had large effects on the substance and energy metabolism and cellular redox balance of C. glutamicum. At low DO, the glycolysis pathway was up-regulated, and TCA was shunted by the up-regulation of the glyoxylate pathway and over-production of amino acids, including valine, cysteine and arginine. Due to the lack of electron-acceptor oxygen, 7 genes related to the electron transfer chain were changed, causing changes in the intracellular ATP content at 0% and 30% DO. The metabolic flux was changed to rebalance the cellular redox. This study applied deep sequencing to identify a wealth of genes and pathways that changed under different DO conditions and provided an overall comprehensive view of the metabolism of C. glutamicum. The results provide potential ways to improve the oxygen tolerance of C. glutamicum and to modify the metabolic flux for amino acid production and heterologous protein expression.

WDR5 Expression Is Prognostic of Breast Cancer Outcome

WDR5 is a core component of the human mixed lineage leukemia-2 complex, which plays central roles in ER positive tumour cells and is a major driver of androgen-dependent prostate cancer cell proliferation. Given the similarities between breast and prostate cancers, we explore the potential prognostic value of WDR5 gene expression on breast cancer survival. Our findings reveal that WDR5 over-expression is associated with poor breast cancer clinical outcome in three gene expression data sets and BreastMark. The eQTL analysis reveals 130 trans-eQTL SNPs whose genes mapped with statistical significance are significantly associated with patient survival. These genes together with WDR5 are enriched with “cellular development, gene expression, cell cycle” signallings. Knocking down WDR5 in MCF7 dramatically decreases cell viability, but does not alter tumour cell response to doxorubicin. Our study reveals the prognostic value of WDR5 expression in breast cancer which is under long-range regulation of genes involved in cell cycle, and anthracycline could be coupled with treatments targeting WDR5 once such a regimen is available.