Juan Zhong

The artificial neural network approach based on uniform design to optimize the fed-batch fermentation condition: application to the production of iturin A.

BackgroundIturin A is a potential lipopeptide antibiotic produced by Bacillus subtilis. Optimization of iturin A yield by adding various concentrations of asparagine (Asn), glutamic acid (Glu) and proline (Pro) during the fed-batch fermentation process was studied using an artificial neural network-genetic algorithm (ANN-GA) and uniform design (UD). Here, ANN-GA based on the UD data was used for the first time to analyze the fed-batch fermentation process. The ANN-GA and UD methodologies were compared based on their fitting ability, prediction and generalization capacity and sensitivity analysis.ResultsThe ANN model based on the UD data performed well on minimal statistical designed experimental number and the optimum iturin A yield was 13364.5 ± 271.3 U/mL compared with a yield of 9929.0 ± 280.9 U/mL for the control (batch fermentation without adding the amino acids). The root-mean-square-error for the ANN model with the training set and test set was 4.84 and 273.58 respectively, which was more than two times better than that for the UD model (32.21 and 483.12). The correlation coefficient for the ANN model with training and test sets was 100% and 92.62%, respectively (compared with 99.86% and 78.58% for UD). The error% for ANN with the training and test sets was 0.093 and 2.19 respectively (compared with 0.26 and 4.15 for UD). The sensitivity analysis of both methods showed the comparable results. The predictive error of the optimal iturin A yield for ANN-GA and UD was 0.8% and 2.17%, respectively.ConclusionsThe satisfactory fitting and predicting accuracy of ANN indicated that ANN worked well with the UD data. Through ANN-GA, the iturin A yield was significantly increased by 34.6%. The fitness, prediction, and generalization capacities of the ANN model were better than those of the UD model. Further, although UD could get the insight information between variables directly, ANN was also demonstrated to be efficient in the sensitivity analysis. The results of these comparisons indicated that ANN could be a better alternative way for fermentation optimization with limited number of experiments.

Isolation of genes related to abscisic acid production in Botrytis cinerea TB‐3‐H8 by cDNA‐AFLP

Abscisic acid (ABA) plays important roles in many aspects of plant growth and development. Botrytis cinerea TB‐3‐H8, a high‐yield strain of ABA, was used to elucidate the molecular mechanisms of ABA production in the present work. cDNA‐amplified fragment length polymorphism (cDNA‐AFLP) technique was applied to isolate genes differentially expressed between ABA high and low‐yield conditions. This resulted in the identification of 856 differentially expressed transcript‐derived fragments (TDFs). Forty‐five TDFs that displayed obvious up‐regulated expression profiles in the ABA high‐yield condition were sequenced. Based on BlastX in NCBI, 31 TDFs were assumed to have homology with genes encoding proteins with known functions. According to molecular function of gene ontology (GO) analysis, the 31 TDFs were categorized to proteins with enzyme catalytic activities, transcription factor activities, transporter activities, and kinds of binding activities. Further confirmation of the differential expression of these sequences was carried out by performing semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) on 10 randomly selected TDFs. Five up‐regulated genes were selected to analyze the expression profiles using real‐time PCR. This study enriches our knowledge of the molecular basis for ABA biosynthesis in B. cinerea TB‐3‐H8.

Comparative transcriptome analysis between an evolved abscisic acid-overproducing mutant Botrytis cinerea TBC-A and its ancestral strain Botrytis cinerea TBC-6

Abscisic acid (ABA) is a classical phytohormone which plays an important role in plant stress resistance. Moreover, ABA is also found to regulate the activation of innate immune cells and glucose homeostasis in mammals. Therefore, this ‘stress hormone’ is of great importance to theoretical research and agricultural and medical applications. Botrytis cinerea is a well-known phytopathogenic ascomycete that synthesizes ABA via a pathway substantially different from higher plants. Identification of the functional genes involved in ABA biosynthesis in B. cinerea would be of special interest. We developed an ABA-overproducing mutant strain, B. cinerea TBC-A, previously and obtained a 41.5-Mb genome sequence of B. cinerea TBC-A. In this study, the transcriptomes of B. cinerea TBC-A and its ancestral strain TBC-6 were sequenced under identical fermentation conditions. A stringent comparative transcriptome analysis was performed to identify differentially expressed genes participating in the metabolic pathways related to ABA biosynthesis in B. cinerea. This study provides the first global view of the transcriptional changes underlying the very different ABA productivity of the B. cinerea strains and will expand our knowledge of the molecular basis for ABA biosynthesis in B. cinerea.

Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis

A high performance capillary electrophoresis (HPCE) method was presented to identify and quantitate free amino acids during fermentation by Bacillus subtilis. Amino acids, pre-column derivatized with phenylisothicyanate, were separated and characterized by HPCE. In order to optimize separation conditions, the assay was developed by varying the β-cyclodextrin concentration and pH of the background electrolyte. A buffer system comprising 30 mM phosphate and 3 mM β-cyclodextrin at pH 7.0, voltage of 20 kV and detection wavelength of 254 nm showed the best results, with 17 out of 20 phenylthioncarbamyl amino acids in a solution adequately separated. For quantification, p-aminobenzoic acid was added as an internal standard. Analysis of free amino acids in Bacillus subtilis culture medium using this method revealed good consistency with the values obtained using conventional ninhydrin-based amino acid analyzer. Four free amino acids (aspartic acid, glutamic acid, proline, and tyrosine) concentration in an extracellular matrix during fermentation by Bacillus subtilis were mainly monitored using this method.

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes

Bacillus subtilis ZK0, which was isolated from cotton, produces a type of lipopeptide antibiotic iturin A that inhibits the growth of pathogenic fungi on agricultural crops. However, the low level of iturin A production by B. subtilis ZK0 does not support its large‐scale application. In this study, B. subtilis ZK0 was subjected to genetic manipulation to improve iturin A production. By the independent or simultaneous overexpression of two regulatory genes (comA and sigA), iturin A production by B. subtilis ZK0 was significantly increased. When both genes were simultaneously overexpressed under optimal conditions, iturin A production increased up to 215 mg l−1 (an approximate 43‐fold increase compared with B. subtilis ZK0). Moreover, overexpression of both genes was unexpectedly found to inhibit biofilm formation by B. subtilis ZK0, indicating that the phenomenon of ‘stuck fermentation’ would be avoided during B. subtilis ZK0 fermentation. In conclusion, a genetic manipulation method that improves iturin A production and inhibits biofilm formation in B. subtilis ZK0 is reported for the first time and this method has the potential to be widely applied in B. subtilis ZK0 commercial fermentation.

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

To introduce DNA into Streptomyces noursei xinao-4, which produces xinaomycins, we explored an intergeneric conjugal transfer system. High efficiency of conjugation (8 × 10−3 exconjugants per recipient) was obtained when spores of S. noursei xinao-4 were heat-shocked at 50 °C for 10 min, mixed with Escherichia coli ET12567 (pUZ8002/pSET152) in the ratio of 1:100, plated on 2CMY medium containing 40 mmol/L MgCl2, and incubated at 30 °C for 22 h. With this protocol, the plasmids pKC1139 and pSET152 were successfully transferred from E. coli ET12567 (pUZ8002) with different frequencies. Among all parameters, the ratio of donor to recipient cell number had the strongest effect on the transformation efficiency. In order to validate the above intergeneric conjugal transfer system, a glycosyltransferase gene was cloned and efficiently knocked out in S. noursei xinao-4 using pSG5-based plasmid pKC1139.

Cys2His2 Zinc Finger Transcription Factor BcabaR1 Positively Regulates Abscisic Acid Production in Botrytis cinerea

Abscisic acid (ABA) could make a potentially important contribution to theoretical research and applications in agriculture and medicine. Botrytis cinerea is a plant-pathogenic fungus that was found to produce ABA. There has been a view that ABA is related to the interaction between pathogenic fungi and plants. Identification of regulatory genes involved in ABA biosynthesis may facilitate an understanding of the underlying molecular mechanisms of ABA biosynthesis and the pathogenesis of B. cinerea. Here, we present a positive regulator, BcabaR1, of ABA biosynthesis in B. cinerea that can affect the transcriptional level of the ABA biosynthesis gene cluster, bcaba1 to -4, by directly binding to the conserved sequence elements in the promoter of the bcaba1 to -4 genes. This TF was found to be specifically involved in regulation of ABA biosynthesis. This work provides new clues for finding other ABA biosynthesis genes and improving ABA yield in B. cinerea. ABSTRACT Abscisic acid (ABA) is one of the five classical phytohormones involved in increasing the tolerance of plants for various kinds of stresses caused by abiotic or biotic factors, and it also plays important roles in regulating the activation of innate immune cells and glucose homeostasis in mammals. For these reasons, as a “stress hormone,” ABA has recently received attention as a candidate drug for agriculture and biomedical applications, prompting significant development of ABA synthesis. Some plant-pathogenic fungi can synthesize natural ABA. The fungus Botrytis cinerea has been used for biotechnological production of ABA. Identification of the transcription factors (TFs) involved in regulation of ABA biosynthesis in B. cinerea would provide new clues to understand how ABA is synthesized and regulated. In this study, we defined a novel Cys2His2 TF, BcabaR1, that regulates the transcriptional levels of ABA synthase genes (bcaba1, bcaba2, bcaba3, and bcaba4) in an ABA-overproducing mutant, B. cinerea TBC-A. Electrophoretic mobility shift assays revealed that recombinant BcabaR1 can bind specifically to both a 14-nucleotide sequence motif and a 39-nucleotide sequence motif in the promoter region of bcaba1 to -4 genes in vitro. A decreased transcriptional level of the bcabaR1 gene in B. cinerea led to significantly decreased ABA production and downregulated transcription of bcaba1 to -4. When bcabaR1 was overexpressed in B. cinerea, ABA production was significantly increased, with upregulated transcription of bcaba1 to -4. Thus, in this study, we found that BcabaR1 acts as a positive regulator of ABA biosynthesis in B. cinerea. IMPORTANCE Abscisic acid (ABA) could make a potentially important contribution to theoretical research and applications in agriculture and medicine. Botrytis cinerea is a plant-pathogenic fungus that was found to produce ABA. There has been a view that ABA is related to the interaction between pathogenic fungi and plants. Identification of regulatory genes involved in ABA biosynthesis may facilitate an understanding of the underlying molecular mechanisms of ABA biosynthesis and the pathogenesis of B. cinerea. Here, we present a positive regulator, BcabaR1, of ABA biosynthesis in B. cinerea that can affect the transcriptional level of the ABA biosynthesis gene cluster, bcaba1 to -4, by directly binding to the conserved sequence elements in the promoter of the bcaba1 to -4 genes. This TF was found to be specifically involved in regulation of ABA biosynthesis. This work provides new clues for finding other ABA biosynthesis genes and improving ABA yield in B. cinerea.