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Doxorubicin is one of the most widely used antitumor drugs and is currently produced via the chemical conversion method, which suffers from high production costs, complex product separation processes, and serious environmental pollution. Biocatalysis is considered a more efficient and environment-friendly method for drug production. The cytochrome daunorubicin C-14 hydroxylase (DoxA) is the essential enzyme catalyzing the conversion of daunorubicin to doxorubicin. Herein, the DoxA from Streptomyces peucetius subsp. caesius ATCC 27952 was expressed in Escherichia coli, and the rational design strategy was further applied to improve the enzyme activity. Eight amino acid residues were identified as the key sites via molecular docking. Using a constructed screening library, we obtained the mutant DoxA(P88Y) with a more rational protein conformation, and a 56% increase in bioconversion efficiency was achieved by the mutant compared to the wild-type DoxA. Molecular dynamics simulation was applied to understand the relationship between the enzyme’s structural property and its substrate-binding efficiency. It was demonstrated that the mutant DoxA(P88Y) formed a new hydrophobic interaction with the substrate daunorubicin, which might have enhanced the binding stability and thus improved the catalytic activity. Our work lays a foundation for further exploration of DoxA and facilitates the industrial process of bio-production of doxorubicin.

Great interest for large-scale production of medicinal mushroom biomass and various pharmaceutically active compounds production dictates the development of comprehensive technologies. Solid state and submerged cultivations in bioreactors represent the most promising technologies for fast and large amount production of medicinal fungi biomass and pharmaceutically active products for human and veterinary need. There are many stages from shaking culture studies to large-scale industrial production. Pilot-scale studies represent the bridge and the balance between the gap of laboratory and industrial scale. Therefore it is not a surprise that most of pilot-scale results and experiences remain uncovered industrial secrets. This chapter is an overview of available engineering achievements in submerged and solid-state cultivation experiences in pilot-scale bioreactors.

A novel bioactive ganoderic acid, 3, 28-dihydroxy-lanosta-8, 24-dien-26-oic-acid (DHLDOA), was recently biosynthesized using a heterologous host Saccharomyces cerevisiae by our group, but its low fermentation titer was an obstacle to large-scale application. In this study, the positive effect of oxygen supply on DHLDOA production by the yeast fermentation in shake-flask was revealed. The highest cell mass with an OD600 value of 27.92 was obtained at initial KLa of 15.6 h−1, and the highest DHLDOA titer of 8.79 mg/L was obtained at initial KLa of 12.6 h−1, which was 1.64- and 1.77-fold that at 4.2 and 15.6 h−1, respectively. Slightly higher pyruvate accumulation, lower citrate level, and adequate precursor pools of DHLDOA anabolism were found to be associated with higher DHLDOA production at initial KLa of 12.6 h−1, in which relatively higher CYP5150L8 and CYP5139G1 activities were also observed. Taken together, the results indicated that oxygen supply caused the global changes of the yeast metabolism, including the DHLDOA biosynthesis. This work will be helpful to further improvement of DHLDOA production by the yeast fermentation.

Terpenoids are a class of high value-added natural products with a variety of biological functions. Genetically engineered microorganisms, such as those of Escherichia coli and Saccharomyces cerevisiae, have merits in producing plant or fungus-derived terpenoids, due to their mature genetic manipulation, simple nutrient demand and fast growth. Oxygen, as a key environmental factor, is particularly important to microbial metabolism and growth, and suitable oxygen supply is viewed as a prerequisite for realizing highly efficient production of terpenoids by engineered microorganisms. In this article, the role of oxygen in regulating terpenoid bioproduction is overviewed from the viewpoints of cellular carbon metabolism, energy metabolism and terpenoid anabolism. Strategies on adjusting oxygen availability to microorganisms, including genetic modification of cellular metabolism related with oxygen utilization, are summarized and discussed, to provide helpful information for further improvement of terpenoid biosynthesis by microbes.

Roles of functional genes in secondary metabolism of mushroom-forming basidiomycetes are rarely clarified due to the difficulty of gene knock-out in them. In Ganoderma lucidum, an antitumor ganoderic acid (GA) producing basidiomycete, two homologs of a Cys2-His2 (C2H2)-type zinc finger transcription factor (CRZ1) - GlCRZ1 and GlCRZ2 were found by bioinformatic analysis. Using a recently developed CRISPR/Cas9 genome editing platform, each gene of the two CRZ1 orthologs (glcrz1 and glcrz2) was disrupted to reveal their function in calcium signaling of GA biosynthesis. Compared to the wild type strain (WT), in which Ca2+ addition enhanced the GA production, disruption of glcrz1 did not hamper the cell growth but abolished the calcium signaling effect on stimulating GA biosynthesis. For the glcrz2 knocked-out strain, both the mycelial growth and GA synthesis were severely attenuated and its GA production declined even with Ca2+ treatment. These results indicated that the calcium signaling via GlCRZ1 and GlCRZ2 were critical in the calcineurin-mediated regulation of GA biosynthesis by G. lucidum. The work may be also helpful to understand the function of CRZ1 in secondary metabolism regulation in other basidiomycetes.

Ganoderic acids (GAs) are oxygenated lanostane-type triterpenoids from the traditional medicinal mushroom Ganoderma lucidum and of significant biological activities. Although a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA) was found to be biosynthesized from lanosterol, further post-modification of HLDOA is yet unclear. In this work, by using HLDOA as the substrate and a crude enzyme from G. lucidum as the biocatalyst, we observed a new peak in liquid chromatography from the reaction system. The product was purified and identified to be 3-oxo-lanosta-8,24-dien-26-oic acid (OLDOA), which may be converted from HLDOA by a putative dehydrogenase of G. lucidum. The work is useful to future manufacture of GAs as well as their biosynthetic pathway elucidation.

Furfural is one of main inhibitors in hemicellulose hydrolysates such as xylose mother liquor, but its positive effect on the production of validamycin-A (VAL-A), a widely used agricultural antibiotic, was interestingly found in fermentation of Streptomyces hygroscopicus 5008. The furfural level in medium up to 1 g/L was effectively converted to furfuryl alcohol and furoic acid by the microorganism. Both intracellular H2O2 level and ValG enzyme activity of the cells were enhanced by furfural addition. Xylose mother liquor medium with supplementation of about 1 g/L furfural could enhance the VAL-A titer by 39 %. This work is helpful to VAL-A fermentation using the hemicellulose hydrolysate.

Ganoderic acids (GAs), a group of highly oxygenated lanostane-type triterpenoids from the traditional Chinese medicinal mushroom Ganoderma lucidum, possessed significant pharmacological activities. Due to the difficulty in its genetic manipulation, low yield, and slow growth of G. lucidum, biosynthesis of GAs in a heterologous host is a promising alternative for their efficient production. Heterologous production of a GA, 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA), was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA to biosynthesize other GAs remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new GA 3,28-dihydroxy-lanosta-8,24-dien-26-oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by UPLC-APCI-HRMS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmids pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR, and hygromycin-resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418-resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work enriches the GA biosynthetic enzyme library, and is helpful to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum. KEY POINTS: • Another P450 gene responsible for GA's post-modification was discovered and identified. • One new GA, DHLDOA, was identified and produced via engineered yeast. • With the balance of the two CYP genes expression, DHLDOA production was significantly improved.

The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

Growing concerns of environmental pollution and fossil resource shortage are major driving forces for bio‐based production of chemicals traditionally from petrochemical industry. Dicarboxylic acids (DCAs) are important platform chemicals with large market and wide applications, and here the recent advances in bio‐based production of straight‐chain DCAs longer than C4 from biological approaches, especially by synthetic biology, are reviewed. A couple of pathways were recently designed and demonstrated for producing DCAs, even those ranging from C5 to C15, by employing respective starting units, extending units, and appropriate enzymes. Furthermore, in order to achieve higher production of DCAs, enormous efforts were made in engineering microbial hosts that harbored the biosynthetic pathways and in improving properties of biocatalytic elements to enhance metabolic fluxes toward target DCAs. Here we summarize and discuss the current advantages and limitations of related pathways, and also provide perspectives on synthetic pathway design and optimization for hyper‐production of DCAs.

Synthetic biology provides a significant platform in creating novel pathways/organisms for producing useful compounds, while it remains a challenge to enhance the production efficiency. Recently we constructed a recombinant Escherichia coli for glutarate production using a synthetic α-ketoacid reduction pathway, in which α-ketoglutarate is reduced to 2-hydroxyglutarate then converted to glutarate. However, the production titer was low, which may be due to 1) oxygen-sensitive nature of 2-hydroxyglutaryl-CoA dehydratase (HgdABC) and 2) limited cell growth in anaerobic cultivation. Therefore, we developed an aerobic-anaerobic two-stage strategy by growing more cells aerobically, then shifting to anaerobic cultivation to ensure the functional HgdABC for glutarate biosynthesis. The two-stage cultivation resulted in higher production of glutarate and other two C5 dicarboxylic acids – glutaconate and 2-hydroxylglutarate than the original anaerobic process. Furthermore, we used an anaerobically-inducible nar promoter to improve the hgdABC expression responding to aerobic-anaerobic shift. Finally, the glutarate, glutaconate and 2-hydroxyglutarate titer was increased about 2, 5 and 3 times, reaching 11.6, 108.8 and 399.5 mg/L, respectively. The work demonstrated an effective strategy for ameliorating α-ketoacid reduction pathway to produce C5 dicarboxylic acids, as well as the potential of integration of bioprocess and metabolic engineering for enhancing chemicals production by an engineered microorganism.

Amino acid decarboxylases catalyze decarboxylation of amino acids into amines that possess wide industrial applications. As key enzymes in biobased production of industrially important amines such as cadaverine, putrescine and β-alanine, lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase have attracted increasing attention. To develop enzyme variants with superior catalytic properties, there is a great need for high-throughput assay of these decarboxylases. Here we report the development of assays based on the color change of pH indicator – chlorophenol red (CPR) or bromothymol blue (BTB) – in decarboxylation reactions, in which one proton was consumed per carboxylic group decarboxylated resulting in an increase in pH. First, two buffer-indicator pairs, 4-morpholineethanesulfonic acid (MES)-CPR and 3-morpholinopropanesulfonic acid (MOPS)-BTB, were chosen on the basis of their similar pKa values at approximately pH 6.0 and 7.0, both of which are physiologically relevant. Next, the effects of buffer strength and indicator concentration on absorbance changes were examined in assay mixtures with NaOH titration, which mimicked proton consumption in decarboxylation reactions. Finally, high-throughput quantification of lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase was achieved using a microplate format. These results suggest that our indicator assay system may have potential applications for screening diverse decarboxylases.

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.

Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coli with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans -enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite α-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans -glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coli resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola . The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.

Antibody-drug conjugates (ADCs), which combine the exquisite specificity of antibodies with the cell-killing ability of cytotoxic drug payloads, have emerged as an attractive means for treating cancers. All (pre)clinical ADCs employ biosynthesized cytotoxins as their ADC payload precursors (APPs). The cost-effective bioproduction of APPs is receiving great interest from both academia and industry. Given the lack of systematic overviews of the topic, we provide the current status of APPs and focus on their state-of-the-art bioproduction strategies, illustrated with typical examples and critical analyses. Challenges in further enhancing the bioproduction efficiency of APPs and other cytotoxins are also discussed. This research has implications for bioprocess and metabolic engineering, systems and synthetic biology, and biopharmaceutical drug discovery, development, and industrialization.

Higher fungi are regarded as promising cell factories for production of bioactive secondary metabolites, but there is a lack of methods of genetic manipulation, such as gene disruption, which hinders the studies on biosynthesis and its regulation of those useful natural products. In this study, the CRISPR-Cas9 assisted gene disruption was established for the first time in higher fungi by taking Ganoderma species as typical examples. With double strand break (DSB) introduced by CRISPR-Cas9, non-homologous end joining (NHEJ) was induced and further assisted the gene disruption. As proof of concept, the ura3 gene of G. lucidum 260125 and G. lingzhi was successfully disrupted by the codon-optimized Cas9 and in vitro transcribed gRNA. This work may help to provide a widely applicable approach of gene disruption in higher fungi.

Microalgae, a group of photosynthetic microorganisms, are a promising feedstock for biodiesel production, but their biomass retrieval is a challenging task. Flocculation is a feasible method for dewatering and harvesting microalgae biomass. In the current study, the effect of alum flocculation on Chlorella vulgaris biomass retrieval has been studied. Alum structural changes with pH were led to a full factorial design to address the effect of this chemical structure changes at different pH values. It is observed that the best flocculation efficiency could be achieved in the natural pH value of C. vulgaris growth medium (8.2) with less than 0.5 g/L flocculant addition, which would lead to the flocculation efficiency of more than 90%. An ensemble architecture of neural networks successfully employed for flocculation modeling.

In this work, the hydrolytic kinetic of piceid was systematically investigated not only in different solvents, but under different conditions of pH and temperature as well. Results showed that the solvents have a significant effect on rate constant. In general, piceid was more readily hydrolyzed in pure organic solvents in which the rate constant was over 10 times of that in conventional aqueous methanol. The solvent effect was analyzed using LSER and it demonstrated that hydrolysis of piceid is accelerated by solvents with larger value of dipolarity/polarizability, being inhibited by hydrogen bond donor solvents. Furthermore, the rate constant increased with elevated acidity and temperature, resulting in shorter time for hydrolysis of piceid and avoiding degradation of resveratrol. The hydrolysis of piceid obeyed first-order kinetics. The new information obtained herein could be helpful to enhanced production of resveratrol via hydrolysis of piceid.

Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer for industrial and biomedical applications. Since farming spiders is not feasible due to their highly territorial nature, recombinant production of dragline silk proteins in a foreign host has received great attention. However, their production titer remains low, because efficient expression of very large, highly repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3–256.5 kDa were significantly elevated by down-shifting the induction temperature. The beneficial effect was found to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combination of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 g l−1 of a 201.6-kDa MaSp2 protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins.

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is an important antitumor agent for cancer treatment, but its market supply suffers from a low production titer. The role of AP-3 unusual glycolate unit supply on its biosynthesis was investigated in this work by overexpressing the responsible gene cluster asm13-17 in A. pretiosum (WT). As a result, the accumulation of AP-3 and its intermediate glyceryl-S-ACP in the asm13-17-overexpressed strain (Oasm13-17) versus WT was enhanced by 1.94 and 1.49-fold, respectively. To provide a higher supply of another precursor 3-amino-5-hydroxybenzoic acid, asmUdpg was also overexpressed in Oasm13-17 (Oasm13-17:asmUdpg), and an improved AP-3 titer of 680.5 mg/L was achieved in shake flasks. To further enhance the AP-3 titer, a rational fed-batch strategy was developed in bioreactor fermentation of Oasm13-17:asmUdpg; and by pulse feeding 15 g/L fructose and 1.64 g/L isobutanol at 60, 96, and 120 hr, the AP-3 production level reached 757.7 mg/L, which is much higher than ever reported in bioreactors. This work demonstrated that a rational approach combining precursor pathway engineering with substrate feeding was very effective in enhancing the AP-3 titer, and this enabling methodology would be helpful to industrial production of this eye-catching drug.

Ganoderma, as a Chinese traditional medicine, has multiple bioactivities. However, industrial production was limited due to low yield during Ganoderma fermentation. In this work, sucrose was found to greatly enhance intracellular polysaccharide (IPS) content and specific extracellular polysaccharide (EPS) production rate. The mechanism was studied by analyzing the activities of enzymes related to polysaccharide biosynthesis. The results revealed that sucrose regulated the activities of phosphoglucomutase and phosphoglucose isomerase. When glucose and sucrose mixture was used as carbon source, biomass, polysaccharide and ganoderic acids (GAs) production was greatly enhanced. A sucrose fed-batch strategy was developed in 10-L bioreactor, and was scaled up to 300-L bioreactor. The biomass, EPS and IPS production was 25.5, 2.9 and 4.8 g/L, respectively, which was the highest biomass and IPS production in pilot scale. This study provides information for further understanding the regulation mechanism of Ganoderma polysaccharide biosynthesis. It demonstrates that sucrose fed-batch is a useful strategy for enhancing Ganoderma biomass, polysaccharide and GAs production.

To further improve the ganoderic acid (GA) production, a novel integrated strategy by combining nitrogen limitation and calcium ion addition was developed. The effects of the integrated combination on the content of GA-T (one powerful anticancer compound), their intermediates (squalene and lanosterol) and on the transcription levels of GA biosynthetic genes in G. lucidum fermentation were investigated. The maximum GA-T content with the integrated strategy were 1.87 mg/ 100 mg dry cell weight, which was 2.1-4.2 fold higher than that obtained with either calcium ion addition or nitrogen limitation alone, and it is also the highest record as ever reported in submerged fermentation of G. lucidum. The squalene content was increased by 3.9- and 2.2-fold in this case compared with either individual strategy alone. Moreover, the transcription levels of the GA biosynthetic genes encoding 3-hydroxy-3-methyglutaryl coenzyme A reductase and lanosterol synthase were also up-regulated by 3.3-7.5 and 1.3-2.3 fold, respectively.

Ganoderic acids (GAs) produced by Ganoderma lucidum possess anticancer activities with the generation of reactive oxygen species (ROS). However, the role of oxidative stress in apoptotic process induced by GAs is still undefined. In this study, the effects of four structurally related GAs, i.e. GA-T, GA-Mk, and two deacetylated derivatives of GA-T (GA-T1 and GA-T2) on the antioxidant defense system and induced apoptosis in cervical cancer cells HeLa were investigated in vitro. Our results indicated that the tested GAs (5-40 mu M) induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9 and caspase-3. Furthermore. GAs increased the generation of intracellular ROS and attenuated antioxidant defense system by decreasing glutathione (GSH) level, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities. The above effects were remarkably blocked by the exogenous antioxidants, i.e. N-acetylcysteine, catalase and diphenyleneiodonium chloride. The potency of the four GAs toward induced apoptosis, generation of ROS and suppression of antioxidant defense system was in the order of: GA-T > GA-Mk approximate to GA-T1 > GA-T2 in HeLa cells. These findings suggest that GAs induced mitochondria-dependent cell apoptosis in HeLa cells are mediated via enhancing oxidative stress and depressing antioxidant defense. Additionally, the acetylation of hydroxyl groups in GAs may contribute to their pro-oxidant activities and cytotoxicity, which is helpful to the development of novel chemotherapy agents. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Commercialization of lignocellulosic ethanol fermentation requires its high titer, but the reactive oxygen species (ROS) accumulation during the bioprocess damaged the cells and compromised this goal. To improve the cellular anti-oxidative activity during non-detoxified corncob residue hydrolysate fermentation, seed cells were prepared to possess a higher level of intracellular biotin pool (IBP), which facilitated the biosyntheses of catalase and porphyrin. As a result, the catalase activity increased by 1.3-folds compared to control while the ROS level reduced by 50%. Cell viability in high-IBP cells was 1.7-folds of control and the final ethanol titer increased from 31.2 to 41.8 g L(-1) in batch fermentation. The high-IBP cells were further used for repeated-batch fermentation in the non-detoxified lignocellulosic hydrolysate, and the highest titer and average productivity of ethanol reached 63.7 g L(-1) and 1.2 g L(-1)h(-1). The results were favorable to future industrial application of this lignocellulosic bioethanol process.

Validamycin A (VAL-A) is an important agricultural antibiotic produced by Streptomyces hygroscopicus 5008, which uses starch as carbon source occupying about 20% of total production cost. To reduce the medium cost, corncob hydrolysate - a hemicellulose hydrolysate was applied as a low-cost substrate to VAL-A fermentation. It was found that three major sugars in corncob hydrolysate including d-glucose, d-xylose and l-arabinose could all be utilized by S. hygroscopicus 5008 to produce VAL-A while d-xylose was the main contributor. A higher VAL-A production titer from d-xylose was achieved by using a genetically engineered strain TC03 derived from S. hygroscopicus 5008, which resulted in 1.27-fold improvement of VAL-A production from the medium containing 13% (v/v) corncob hydrolysate compared to that by its original strain. A medium cost analysis was done and compared with previous reports. This work indicates a great potential of the hemicellulose hydrolysate as substrate for antibiotic fermentation.

Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of cytochrome P450 monooxygenase (CYP450) gene candidates from G. lucidum, which may be responsible for the GA biosynthesis from lanosterol but have not been functionally characterized. As a result, overexpression of a CYP450 gene cyp5150l8 was firstly found to produce an antitumor GA, 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) in S. cerevisiae, as confirmed by HPLC, LC-MS and NMR. A final titer of 14.5 mg/L of HLDOA was obtained at 120 hr of the yeast fermentation. Furthermore, our in vitro enzymatic experiments indicate that CYP5150L8 catalyzes a three-step biotransformation of lanosterol at C-26 to synthesize HLDOA. To our knowledge, this is the first report on the heterologous biosynthesis of GAs. The results will be helpful to the GA biosynthetic pathway elucidation and to future optimization of heterologous cell factories for GA production.

Understanding the hierarchical structure of proteins at their fundamental length scales is essential to get insights into their functions and roles in fundamental biological processes. Near-field scanning optical microscopy (NSOM), which overcomes the diffraction limits of conventional optics, provides a powerful analytical tool to image target proteins at nanoscale resolution. Especially, by combining NSOM with infrared (IR) or Raman spectroscopy, near-field nanospectroscopic imaging of a single protein is achieved. In this review, we present the recent technical progress of NSOM setup for nanospectroscopic imaging of proteins, and its application to nanospectroscopic analysis of protein structures is highlighted and critically reviewed. Finally, current challenges and perspectives on application of NSOM in emerging areas of industrial, environmental and medical biotechnology are discussed.

A bioelectrochemical sensing system based on a novel whole-cell redox reactivation/cycling module for ultrasensitive detection of pyocyanin (the biomarker of Pseudomonas aeruginosa infections) was developed. The electroactive bacteria mediated redox reactivation module was constructed using Shewanella oneidensis MR-1 cells as the bioelectro-catalyst and lactate as the electron donor. It could regenerate reductive pyocyanin from its oxidative state, which enabled pyocyanin molecule repeatedly registered by the electrode. Uniquely, with this redox reactivation module, the electrochemical response of pyocyanin was amplified about 405 times (1.3 μA/nM vs. 3.2nA/nM). Thus, an ultrasensitive bioelectrochemical sensing system for pyocyanin quantification was developed by integrating the pyocyanin reactivation module with conventional electrochemical detection system. Remarkably, with this developed biosensing system, an extremely low LOD of 47±1pM was reached. Additionally, this biosensing system showed excellent resistance to interferences from human fluids or bacterial contamination. This work provided a simple, ultrasensitive and robust tool for pyocyanin detection, and more importantly, demonstrated a new dimension for electrochemical signal amplification in biosensing.

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression.

Two-stage cultivation of Ganoderma lucidum was performed for the enhanced production of ganoderic acid (GA). Cellulase was identified to be an effective elicitor for the improvement of GA production, and GA titer reached 1334.5 mg/l compared to the control (779.6 mg/l) using lactose as the substrate without cellulase addition. Loading of 5 mg/l cellulase on day 3 resulted in the maximal GA titer of 1608 mg/l. To our knowledge, this is the first time that cellulase was used as the elicitor to enhance GA production. Submerged fermentation in a 2.0-l bioreactor was also conducted with cellulase as the elicitor, and as a result the maximal GA titer of 1252.7 mg/l was obtained on day 12. This is so far the best GA production obtained in submerged fermentation of G . lucidum .

Metal ions affect cell physiology and metabolism significantly, but the role of Mn(2+) in the secondary metabolism of mushrooms is yet unclear. In static liquid cultivation of Ganoderma lucidum for producing antitumor ganoderic acids (GAs), the Mn(2+) addition was performed. Addition of 10 mM Mn(2+) at the start of the static liquid cultivation resulted in 2.2-fold improvement of total GAs production. The expression levels of GA biosynthetic and Ca(2+) sensors' genes were up-regulated with Mn(2+) induction while down-regulated by adding cyclosporin A (calcineurin inhibitor), suggesting that higher GA production might result from calcineurin signal regulation. Intracellular Ca(2+) imaging and calcineurin inhibitor study revealed that addition of Mn(2+) led to Ca(2+) influx from medium to the cells to trigger calcineurin signals. Mn(2+) addition was therefore an efficient induction strategy for improving GAs production, whose regulation mechanism was via calcineurin signaling transduction.

The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production.

Pichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8 , which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii . This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8 -disrupted (Δ CAT8 ) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the Δ CAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4 , a transcriptional respiratory activator, was significantly repressed in the Δ CAT8 strain. Through disruption of HAP4 , the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the Δ CAT8 strain. A further transcriptional comparison between Δ CAT8 and Δ HAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4 . The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts.

Rapid and efficient extraction of bioactive compounds from biomass for sample analysis presents one of the major challenges in high-productive strain screening and bioprocess development. In this work, a high-throughput method based on a new setup was developed for the extraction of β-carotene from Blakeslea trispora. After the cell was completely disrupted by high-throughput machinery grinding, β-carotene was completely extracted by chloroform and dimethylsulfoxide (1:1, v/v). According to the experimental results, the newly developed high-throughput extraction (HTPE) method could simultaneously process 96 samples within 30 Min, and only 5-mL solvent was used for each sample. The sufficient extraction of β-carotene by the HTPE method, which was confirmed by the conventional reference extraction method, further demonstrated that the newly developed HTPE method was more efficient and economical than the methods developed previously for the extraction of β-carotene.

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett-Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.

Lavendamycin methyl ester (LME) is a derivative of a highly functionalized aminoquinone alkaloid lavendamycin and could be used as a scaffold for novel anticancer agent development. This work demonstrated LME production by cultivation of an engineered strain of Streptomyces flocculus CGMCC4.1223 ΔstnB1, while the wild-type strain did not produce. To enhance its production, the effect of shear stress and oxygen supply on ΔstnB1 strain cultivation was investigated in detail. In flask culture, when the shaking speed increased from 150 to 220 rpm, the mycelium was altered from a large pellet to a filamentous hypha, and the LME production was almost doubled, while no significant differences were observed among varied filling volumes, which implied a crucial role of shear stress in the morphology and LME production. To confirm this suggestion, experiments with agitation speed ranging from 400 to 1,000 rpm at a fixed aeration rate of 1.0 vvm were conducted in a stirred tank bioreactor. It was found that the morphology became more hairy with reduced pellet size, and the LME production was enhanced threefolds when the agitation speed increased from 400 to 800 rpm. Further experiments by varying initial k L a value at the same agitation speed indicated that oxygen supply only slightly affected the physiological status of ΔstnB1 strain. Altogether, shear stress was identified as a major factor affecting the cell morphology and LME production. The work would be helpful to the production of LME and other secondary metabolites by filamentous microorganism cultivation.

To confront energy shortage, global warming and climate changes, biofuels derived from biomass have received increasing attention from the industry, academia and governments. Of the potential sources of biofuels a most promising one is the simple photosynthetic microalgae, which can be grown in open ponds, photobioreactors and fermenters. The advantages to produce biofuels from microalgae include easy adaption to environmental conditions, high photosynthesis efficiency, high lipid content and noncompetition for farmlands. Nonetheless, the real hallmark of microalgae is the fact that these microscopic organisms can provide the biomass feedstock for the flexible production of several different types of renewable and sustainable biofuels such as biodiesel, bioethanol, biogas, biohydrogen among others via thermochemical and biochemical conversion processes. Amazingly, from a sustainability perspective the integrated algal biofuels production, where biodiesel, bioethanol and biogas are continuously produced from one biomass source, can evidently lead to an increase in the energetic productivity of the microalgal biomass, thus improving the economics of this algal biorefinery approach. Developments in several areas, such as genetic and metabolic engineering, are expected to further improve the costeffectiveness of the biofuels from microalgae in an environmentally sustainable manner.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the β cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into β cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.