Identification of a novel growth-associated promoter for biphasic expression of heterogenous proteins in Pichia pastoris.

Pichia pastoris (P. pastoris) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (PAOX1) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for PAOX1, methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (PFDH800). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the PFDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and PAOX1, respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by PFDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of PAOX1. Our PFDH800-based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the PAOX1 promoter.IMPORTANCEThe alcohol oxidase 1 promoter (PAOX1) expression system has the characteristics of biphasic expression and high expression levels, making it the most widely used promoter in the yeast Pichia pastoris. However, PAOX1 requires methanol induction, which can be toxic and poses a fire hazard in large quantities. Our research has found that the activity of PFDH800 is closely related to the growth state of cells and can achieve biphasic expression without the need for an inducer. Compared to other reported non-methanol-induced biphasic expression systems, the system based on the PFDH800 offers several advantages, including high expression levels, simple construction, minimal impact on cellular metabolism, no need for an inducer, and the ability to fine-tune expression.

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Genomic alterations driving precancerous to cancerous lesions in esophageal cancer development.

Esophageal squamous cell carcinoma (ESCC) develops through a series of increasingly abnormal precancerous lesions. Previous studies have revealed the striking differences between normal esophageal epithelium and ESCC in copy number alterations (CNAs) and mutations in genes driving clonal expansion. However, due to limited data on early precancerous lesions, the timing of these transitions and which among them are prerequisites for malignant transformation remained unclear. Here, we analyze 1,275 micro-biopsies from normal esophagus, early and late precancerous lesions, and esophageal cancers to decipher the genomic alterations at each stage. We show that the frequency of TP53 biallelic inactivation increases dramatically in early precancerous lesion stage while CNAs and APOBEC mutagenesis substantially increase at late stages. TP53 biallelic loss is the prerequisite for the development of CNAs of genes in cell cycle, DNA repair, and apoptosis pathways, suggesting it might be one of the earliest steps initiating malignant transformation.

Collagen 1-mediated CXCL1 secretion in tumor cells activates fibroblasts to promote radioresistance of esophageal cancer.

Esophageal squamous-cell carcinoma (ESCC) is commonly treated with radiotherapy; however, radioresistance hinders its clinical effectiveness, and the underlying mechanism remains elusive. Here, we develop patient-derived xenografts (PDXs) from 19 patients with ESCC to investigate the mechanisms driving radioresistance. Using RNA sequencing, cytokine arrays, and single-cell RNA sequencing, we reveal an enrichment of cancer-associated fibroblast (CAF)-derived collagen type 1 (Col1) and tumor-cell-derived CXCL1 in non-responsive PDXs. Col1 not only promotes radioresistance by augmenting DNA repair capacity but also induces CXCL1 secretion in tumor cells. Additionally, CXCL1 further activates CAFs via the CXCR2-STAT3 pathway, establishing a positive feedback loop. Directly interfering with tumor-cell-derived CXCL1 or inhibiting the CXCL1-CXCR2 pathway effectively restores the radiosensitivity of radioresistant xenografts in vivo. Collectively, our study provides a comprehensive understanding of the molecular mechanisms underlying radioresistance and identifies potential targets to improve the efficacy of radiotherapy for ESCC.

Refined expression quantitative trait locus analysis on adenocarcinoma at the gastroesophageal junction reveals susceptibility and prognostic markers.

Objectives: This study aimed to explore cell type level expression quantitative trait loci (eQTL) in adenocarcinoma at the gastroesophageal junction (ACGEJ) and identify susceptibility and prognosis markers. Methods: Whole-genome sequencing (WGS) was performed on 120 paired samples from Chinese ACGEJ patients. Germline mutations were detected by GATK tools. RNA sequencing (RNA-seq) data on ACGEJ samples were taken from our previous studies. Public single-cell RNA sequencing (scRNA-seq) data were used to produce the proportion of epithelial cells. Matrix eQTL and a linear mixed model were used to identify condition-specific cis-eQTLs. The R package coloc was used to perform co-localization analysis with the public data of genome-wide association studies (GWASs). Log-rank and Cox regression tests were used to identify survival-associated eQTL and genes. Functions of candidate risk loci were explored by experimental validation. Results: Refined eQTL analyses of paired ACGEJ samples were performed and 2,036 potential ACGEJ-specific eQTLs with East Asian specificity were identified in total. ACGEJ-gain eQTLs were enriched at promoter regions more than ACGEJ-loss eQTLs. rs658524 was identified as the top eQTL close to the transcription start site of its paired gene (CTSW). rs2240191-RASAL1, rs4236599-FOXP2, rs4947311-PSORS1C1, rs13134812-LOC391674, and rs17508585-CDK13-DT were identified as ACGEJ-specific susceptibility eQTLs. rs309483-LINC01355 was associated with the overall survival of ACGEJ patients. We explored functions of candidate eQTLs such as rs658524, rs309483, rs2240191, and rs4947311 by experimental validation. Conclusion: This study provides new risk loci for ACGEJ susceptibility and effective disease prognosis biomarkers.

Comprehensive Analyses Reveal Effects on Tumor Immune Infiltration and Immunotherapy Response of APOBEC Mutagenesis and Its Molecular Mechanisms in Esophageal Squamous Cell Carcinoma.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) mutagenesis is prevalent in esophageal squamous cell carcinoma (ESCC). However, the functional role of APOBEC mutagenesis has yet to be fully delineated. To address this, we collect matched multi-omics data of 169 ESCC patients and evaluate characteristics of immune infiltration using multiple bioinformatic approaches based on bulk and single-cell RNA sequencing (scRNA-seq) data and verified by functional assays. We find that APOBEC mutagenesis prolongs overall survival (OS) of ESCC patients. The reason for this outcome is probably due to high anti-tumor immune infiltration, immune checkpoints expression and immune related pathway enrichment, such as interferon (IFN) signaling, innate and adaptive immune system. The elevated AOBEC3A (A3A) activity paramountly contributes to the footprints of APOBEC mutagenesis and is first discovered to be transactivated by FOSL1. Mechanistically, upregulated A3A exacerbates cytosolic double-stranded DNA (dsDNA) accumulation, thus stimulating cGAS-STING pathway. Simultaneously, A3A is associated with immunotherapy response which is predicted by TIDE algorithm, validated in a clinical cohort and further confirmed in mouse models. These findings systematically elucidate the clinical relevance, immunological characteristics, prognostic value for immunotherapy and underlying mechanisms of APOBEC mutagenesis in ESCC, which demonstrate great potential in clinical utility to facilitate clinical decisions.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g-1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L-1L-met and 0.60 g L-1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L-1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L-1 to 10.1 g L-1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Research progress of L-aspartate-α-decarboxylase and its isoenzyme in the ß-alanine synthesis.

Driven by the massive demand in recent years, the production of ß-alanine has significantly progressed in chemical and biological ways. Although the chemical method is relatively mature compared to biological synthesis, its high cost of waste disposal and environmental pollution does not meet the environmental protection standard. Hence, the biological method has become more prevalent as a potential alternative to the chemical synthesis of ß-alanine in recent years. As a result, the aspartate pathway from L-aspartate to ß-alanine (the most significant rate-limiting step in the ß-alanine synthesis) catalyzed by L-aspartate-α-decarboxylase (ADC) has become a research hotspot in recent years. Therefore, it is vital to comprehensively understand the different enzymes that possess a similar catalytic ability to ADC. This review will investigate the exploratory process of unique synthesis features and catalytic properties of ADC/ADC-like enzymes in particular creatures with similar catalytic capacity or high sequence homology. At the same time, we will discuss the different ß-alanine production methods which can apply to future industrialization.

Characterization of genomic alterations and neoantigens and analysis of immune infiltration identified therapeutic and prognostic biomarkers in adenocarcinoma at the gastroesophageal junction.

Objectives: Adenocarcinoma at the gastroesophageal junction (ACGEJ) refers to a malignant tumor that occurs at the esophagogastric junction. Despite some progress in targeted therapies for HER2, FGFR2, EGFR, MET, Claudin 18.2 and immune checkpoints in ACGEJ tumors, the 5-year survival rate of patients remains poor. Thus, it is urgent to explore genomic alterations and neoantigen characteristics of tumors and identify CD8+ T-cell infiltration-associated genes to find potential therapeutic targets and develop a risk model to predict ACGEJ patients' overall survival (OS). Methods: Whole-exome sequencing (WES) was performed on 55 paired samples from Chinese ACGEJ patients. Somatic mutations and copy number variations were detected by Strelka2 and FACETS, respectively. SigProfiler and SciClone were employed to decipher the mutation signature and clonal structure of each sample, respectively. Neoantigens were predicted using the MuPeXI pipeline. RNA sequencing (RNA-seq) data of ACGEJ samples from our previous studies and The Cancer Genome Atlas (TCGA) were used to identify genes significantly associated with CD8+ T-cell infiltration by weighted gene coexpression network analysis (WGCNA). To construct a risk model, we conducted LASSO and univariate and multivariate Cox regression analyses. Results: Recurrent MAP2K7, RNF43 and RHOA mutations were found in ACGEJ tumors. The COSMIC signature SBS17 was associated with ACGEJ progression. CCNE1 and VEGFA were identified as putative CNV driver genes. PI3KCA and TP53 mutations conferred selective advantages to cancer cells. The Chinese ACGEJ patient neoantigen landscape was revealed for the first time, and 58 potential neoantigens common to TSNAdb and IEDB were identified. Compared with Siewert type II samples, Siewert type III samples had significant enrichment of the SBS17 signature, a lower TNFRSF14 copy number, a higher proportion of samples with complex clonal architecture and a higher neoantigen load. We identified 10 important CD8+ T-cell infiltration-related Hub genes (CCL5, CD2, CST7, GVINP1, GZMK, IL2RB, IKZF3, PLA2G2D, P2RY10 and ZAP70) as potential therapeutic targets from the RNA-seq data. Seven CD8+ T-cell infiltration-related genes (ADAM28, ASPH, CAMK2N1, F2R, STAP1, TP53INP2, ZC3H3) were selected to construct a prognostic model. Patients classified as high risk based on this model had significantly worse OS than low-risk patients, which was replicated in the TCGA-ACGEJ cohort. Conclusions: This study provides new neoantigen-based immunotherapeutic targets for ACGEJ treatment and effective disease prognosis biomarkers.

Multi-omic characterization of genome-wide abnormal DNA methylation reveals diagnostic and prognostic markers for esophageal squamous-cell carcinoma.

This study investigates aberrant DNA methylations as potential diagnosis and prognosis markers for esophageal squamous-cell carcinoma (ESCC), which if diagnosed at advanced stages has <30% five-year survival rate. Comparing genome-wide methylation sites of 91 ESCC and matched adjacent normal tissues, we identified 35,577 differentially methylated CpG sites (DMCs) and characterized their distribution patterns. Integrating whole-genome DNA and RNA-sequencing data of the same samples, we found multiple dysregulated transcription factors and ESCC-specific genomic correlates of identified DMCs. Using featured DMCs, we developed a 12-marker diagnostic panel with high accuracy in our dataset and the TCGA ESCC dataset, and a 4-marker prognostic panel distinguishing high-risk patients. In-vitro experiments validated the functions of 4 marker host genes. Together these results provide additional evidence for the important roles of aberrant DNA methylations in ESCC development and progression. Our DMC-based diagnostic and prognostic panels have potential values for clinical care of ESCC, laying foundations for developing targeted methylation assays for future non-invasive cancer detection methods.

Dissecting esophageal squamous-cell carcinoma ecosystem by single-cell transcriptomic analysis.

Esophageal squamous-cell carcinoma (ESCC), one of the most prevalent and lethal malignant disease, has a complex but unknown tumor ecosystem. Here, we investigate the composition of ESCC tumors based on 208,659 single-cell transcriptomes derived from 60 individuals. We identify 8 common expression programs from malignant epithelial cells and discover 42 cell types, including 26 immune cell and 16 nonimmune stromal cell subtypes in the tumor microenvironment (TME), and analyse the interactions between cancer cells and other cells and the interactions among different cell types in the TME. Moreover, we link the cancer cell transcriptomes to the somatic mutations and identify several markers significantly associated with patients' survival, which may be relevant to precision care of ESCC patients. These results reveal the immunosuppressive status in the ESCC TME and further our understanding of ESCC.

VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

Radiotherapy remains the mainstay for treatment of various types of human cancer; however, the clinical efficacy is often limited by radioresistance, in which the underlying mechanism is largely unknown. Here, using esophageal squamous cell carcinoma (ESCC) as a model, we demonstrate that guanine nucleotide exchange factor 2 (VAV2), which is overexpressed in most human cancers, plays an important role in primary and secondary radioresistance. We have discovered for the first time that VAV2 is required for the Ku70/Ku80 complex formation and participates in non-homologous end joining repair of DNA damages caused by ionizing radiation. We show that VAV2 overexpression substantially upregulates signal transducer and activator of transcription 1 (STAT1) and the STAT1 inhibitor Fludarabine can significantly promote the sensitivity of radioresistant patient-derived ESCC xenografts in vivo in mice to radiotherapy. These results shed new light on the mechanism of cancer radioresistance, which may be important for improving clinical radiotherapy.

Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose.

The miglitol intermediate, 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), is catalyzed from N-2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans/pBBR1-sldAB-pqqABCDE-tldD (G. oxydans/AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans/AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans/AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans/AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans/AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%.

Combinational expression of D-sorbitol dehydrogenase and pyrroloquinoline quinone increases 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production by Gluconobacter oxydans through cofactor manipulation.

6-(N-hydroxyethyl)-amino-6-deoxy-l-sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ±â€¯3.0 g/L of 6NSL production with 94.5 ±â€¯3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess.

Prevention and treatment system of novel coronavirus infection in medical and health institutions: experience in West China Hospital of Sichuan University.

The novel coronavirus infection broke out in Wuhan, China, in December 2019, and progressed to a global pandemic. We describe the measures taken by West China Hospital of Sichuan University to address the diagnosis, prevention and treatment of the infection.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

Repeated production of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose by immobilized Gluconobacter oxydans cells with a strategy of in situ exhaustive cell regeneration.

The major troubles in 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) production from N-2-hydroxyethyl glucamine (NHEG) by Gluconobacter oxydans were low cell yield during cell preparation and loss of cells' biocatalytic ability during biotransformation, resulting in high production cost and low 6NSL production. The target of this work was to enhance 6NSL production by reusing cells and improving the cells biocatalytic ability. First, inhibitory effects of substrate and product on 6NSL production, and optimization of cell regeneration condition were investigated, respectively. Then repeated production of 6NSL by immobilized cell using a strategy of in situ exhaustive cell regeneration in a bubble column bioreactor was developed. As a result, the bioprocess underwent nine cycles, the average 6NSL production and conversion rate of NHEG to 6NSL reached 42.6 g L-1 and 83.1% in each batch was achieved, respectively.

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source.

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9 ± 43.8 mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose.

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L-1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

Integrated strategy of temperature shift and mannitol feeding for enhanced production of echinocandin B by Aspergillus nidulans CCTCC M2012300.

The production of echinocandin B (ECB) by Aspergillus nidulans CCTCC M2012300 was improved by integrating the temperature-shift and fed-batch control strategies. The kinetic characteristics of batch cultures were analyzed at different culture temperatures, and then a two-stage temperature control strategy was established. In the first 6 days, the temperature was maintained at 30 °C to obtain the maximal cell growth rate; subsequently, 25 °C was used to gain a high ECB formation rate. On the basis of temperature control, the ECB productivity was increased to 143.3 mg/(L day), which was a 1.3-fold improvement compared with the optimal constant-temperature cultivations. The influences of fed-batch cultures were further investigated. A maximal ECB productivity of 170.8 mg/(L day) was obtained through a three-stage mannitol pulse-feeding strategy, which was another 1.2-fold improvement than that of the batch fermentation. This is the first report of the use of a two-stage temperature control fed-batch strategy in ECB fermentation. This strategy was simple and economical to operate and may provide new guidance for the industrial-scale production of ECB.