Abstract:Industrial biotechnology, which employs microorganisms or enzymes to produce industrial useful products, has been considered as a promising solution for the sustainable development of society and economy. This special issue collects some recent research progresses on industrial biotechnology in China, including reviews and research articles in the field of genetic engineering, metabolic engineering and synthetic biology, physiological engineering, fermentation engineering and biochemical engineering, biocatalysis and biotransformation, as well as new biotechniques and methods.

Abstract:NB-C1 gene is a potential class IIa bacteriocin gene. To obtain its soluble expression, NB-C1 was fused with the green fluorescent protein (GFP) gene and a recombinant expression vector pIVEX 2.4d-GFP-NB-C1 was constructed, which was transformed into Escherichia coli BL21(DE3) pLysS. The expressed fusion protein GFP-NB-C1 was purified by Ni-NTA affinity chromatography and the bioactivity was examined using Listeria monocytogenes as the indicator bacteria. The results showed that the expressed fusion protein GFP-NB-C1 was soluble and the final concentration of the purified fusion protein was 36.1 mg/L E. coli culture and had the purity above 95%. The antimicrobial assay of GFP-NB-C1 was analyzed and showed its high activity against Listeria monocytogenes.

Abstract:In order to make a large-scale preparation of (GLP-1A2G)2-HAS (GGH), the double-plamid pPICZαB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZαB-ggh, which was transformed into P. pastoris GS115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 °C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.

Abstract:Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtI/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid pAC-LYC△(crtB) carrying crtI/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.

Abstract:Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.DphaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.DphaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.DphaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.DphaC&E was obviously increased. On the third day, the NADPH content in S.DphaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.DphaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.

Abstract:Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L·h).

Abstract:N-Acetylornithine aminotransferase (EC 2.6.1.11, ACOAT) catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine, the forth step involved in the L-arginine biosynthetic pathways. We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization. ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5. Analysis of argD sequences revealed that only one ORF existed, which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa. The argD gene from C. crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C. crenatum SYPA. Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g. Subsequently, the expression plasmid pJCtac-CcargD was transformed into C. crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C. crenatum CCD. Further fermentative character of CCD1 was also analyzed. The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.

Abstract:In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.

Abstract:Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.

Abstract:Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.

Abstract:Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 °C, 3 °C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 °C and 52 °C for 1 h respectively. At temperature higher than 37 °C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 °C to 40 °C, recombinant strain T44-2 produces 91.2–83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

Abstract:Fermentation and induction conditions for recombinant Escherichia coli expressing Thermobifida fusca cutinase were optimized in flasks and 3L fermenter. Surface modification of poly (ethylene terephthalate) fibers with cutinase was also discussed. The results showed that, cutinase yield reached 128 U/mL by adding 2 g/L inducer lactose and 0.5% glycine. In the fed-batch culture in a 3 L fermenter, the maximum biomass cutinase activity was up to 506 U/mL, which is the highest bacterial cutinase activity reported by far. Recombinant cutinase was used to modify polyester fibers and terephthalic acid substance was detected by using UV analysis. The dyeing and wetting properties of cutinase treated fibers were higher than untreated fibers. Combined utilization of cutinase and Triton X-100 can significantly improve the hydrophilicity of polyester. This is the first report of surface modification on polyester fibers by bacterial cutinase.

Abstract:We evaluated an astaxanthin overproducing Phaffia rhodozyma JMU-MVP14, and developed astaxanthin high-yielding fermentation process. We analyzed several fermentation parameters, i.e., biomass, astaxanthin and total carotenoids content to compare the characteristics of P. rhodozyma JMU-MVP14 and the original strain through flask fermentation experiments. We conducted batch and fed-batch fermentation experiments in 7 L fermentor to investigate the effects of pH controlling models and feeding medium compositions on the production of astaxanthin. We further evaluated the capability and practical value of P. rhodozyma JMU-MVP14 by fed-batch cultivation in the 1 m3 fermentor. Flask fermentation experiments revealed that P. rhodozyma JMU-MVP14 produced high yield of astaxanthin and carotenoids with specific productivity of astaxanthin and specific productivity of total carotenoids of 6.01 mg/g and 10.38 mg/g. Results of batch culture experiments in the 7 L fermentor showed that controlling the pH by ammonia auto-feeding was better than discontinuously adjusting pH value at 6.0 with regard to the high productivities of biomasses and astaxanthin. This P. rhodozyma strain synthesized astaxanthin partially linked to the growth with the Ks and μmax of 0.20 h?1 and 21.73 g/L, respectively. Results of batch-fed fermentations in 7 L fermentor indicated that the complex feeding medium consisted of 50% glucose, 0.5% yeast extract and 0.3% corn steep syrup had lower astaxanthin productivity than the simple feeding medium containing only 50% glucose, which produced biomass, volumetric productivity of astaxanthin, volumetric productivity of total carotenoids, specific productivity of astaxanthin and total carotenoids at 32.81 g/L, 155.99 mg/L, 4.94 mg/g, 399.99 mg/L and 12.19 mg/g, respectively. As fed-batch cultured in 1 m3 fermentor, P. rhodozyma JMU-MVP14 yielded 85.11 g/L of biomass, 279.96 mg/L of volumetric productivity of astaxanthin, 618.01 mg/L of volumetric productivity of total carotenoids, 3.29 mg/g of specific productivity of astaxanthin and 7.26 mg/g of specific productivity of total carotenoids. Additionally, P. rhodozyma JMU-MVP14 cell contained 21.54% of protein, 41.34% of carbohydrate and 34.31% of lipid. These comprehensive results suggest that P. rhodozyma JMU-MVP14 has great practical prosperity related to its strong ability to produce astaxanthin and good value byproducts.

Abstract:Fe (III) modified collagen fibers were used to immobilize catalase through the cross-linking of glutaraldehyde. The loading amount of catalase on the supporting matrix was 16.7 mg/g, and 35% enzymatic activity was remained. A series of experiments were conducted on free and immobilized catalase in order to investigate their optimal pH and temperature, and the thermal, storage and operation stability. Results suggest that the free and immobilized catalase prefer similar pH and temperature condition, which were pH 7.0 and 25 °C. It should be noted that the thermal stability of catalase was considerably improved after immobilization owing to the fact that the enzyme kept 30% of relative activity after incubation at 75 °C for 5 h. On the contrary, the free catalase was completely inactive. As for the storage stability, the immobilized catalase kept 88% of relative activity after stored at room temperature for 12 days while the free one was completely inactive under the same conditions. Moreover, the immobilized catalase preserved 57% of relative activity after being reused 26 times, exhibiting excellent operation stability. Consequently, this investigation suggests that collagen fiber can be used as excellent supporting matrix for the immobilization of catalase, and it is potential to be used for the immobilization of similar enzymes.

Abstract:In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl-1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.

Abstract:In order to explore the influence of reaction temperature on the product composition, the effect of continuous temperature change (22 °C?60 °C, ±0.1 °C) on hydrolysis of yeast β-glucan by endo-β-1,3-glucanase was determined by using self-developed Biochem-temperature Characteristic Apparatus. The activation energy of enzymatic hydrolysis of yeast β-glucan was 84.17 kJ/mol. The optimum temperature represented by accumulation of products decreased exponentially within a certain period of time. The components of the products were changed with reaction temperature. The length of oligosaccharides decreased with the increase of temperature. The main products were laminaribiose and laminaritriose at the temperature higher than 46 °C, while the main products were laminaripentaose and larger molecular weight components at the temperature lower than 30 °C. The results can provide precise parameters to control the reaction temperature of the production of 1,3-β-D-glucooligosaccharides.

Abstract:The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 104. Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 °C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471Ile. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Val596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.

Abstract:To identify the anti-bacterial compound(s) from Streptomyces hygroscopicus 17997, a geldanamycin producer, silica gel thin layer chromatography (TLC) TLC was used to separate the secondary metabolites of S. hygroscopicus 17997. Compound(s) from the silica gel TLC with anti-Gram positive bacteria activity and becoming red upon color reaction by 2.0 mol/L NaOH was analyzed by HPLC. The UV absorption profile and the retention time of a peak of HPLC were identical to those of authentic elaiophylin. A conserved region of dTDP-glucose-4,6-dehydratase (Tgd) gene was amplified by PCR from the genomic DNA of Streptomyces hygroscopicus 17997. DNA sequence analysis of the amplified DNA fragment indicated that it should be the tgd gene of elaiophylin biosynthetic gene cluster. These results implied that the compound in the peak of HPLC was elaiophylin, a macrodiolide antibiotic. The compound was then confirmed to be elaiophylin by LC-(+)-ESI-MS, which revealed that Streptomyces hygroscopicus 17997 was an elaiophylin producer. At the same time, a fast procedure, which consisted of silica gel TLC, color reaction, HPLC, PCR detection and DNA sequence analysis of tgd gene, and LC-(+)-ESI-MS, was established for rapid identification of elaiophylin and its producer.