阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

Genome shuffling and high-throughput screening of <Emphasis Type="Italic">Brevibacterium flavum</Emphasis> MDV1 for enhanced <Emphasis Type="SmallCaps">l</Emphasis>-valine production

l-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for l-valine grows rapidly, there is an increasing interest to develop an efficient l-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield l-valine strains to replace the traditional l-valine assay. l-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six l-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an l-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of l-valine (0.598 mol l-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m³ fermentor, this strain produced?>?66.8 g/L l-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of l-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.     

Nisin高产菌株的高通量筛选

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。     

Calcium malate overproduction by Penicillium viticola 152 using the medium containing corn steep liquor

In this study, after screening of eight fungal strains for their ability to produce calcium malate, it was found that Penicillium viticola 152 isolated from marine algae among them could produce the highest titer of calcium malate. At the same time, it was found that corn steep liquor (CSL) could stimulate calcium malate production and 0.5 % (v/v) CSL was the most suitable for calcium malate production. Under the optimal conditions, a titer of calcium malate in the supernatant was 132 g/l at flask level. During a 10-l fermentation, a titer of 168 g/l, a yield of 1.28 g/g of glucose, and a productivity of 1.75 g/l/h were reached within 96 h of the fermentation, and 93.4 % of the sugar was used for calcium malate production and cell growth, demonstrating that the titer, yield, and productivity of calcium malate by this fungal strain were very high and the fermentation period was very short. After analysis of the partially purified product with high-performance liquid chromatography, it was found that the main product was calcium malate. The results demonstrated that P. viticola 152 obtained in this study was the most suitable for developing a novel one-step fermentation process for calcium malate production from glucose on a large scale.     

Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid

In this study, citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 was investigated. After the compositions of the extract of Jerusalem artichoke tubers for citric acid production were optimized, the results showed that natural components of extract of Jerusalem artichoke tubers without addition of any other components were suitable for citric acid production by the yeast strain. During 10 L fermentation using the extract containing 84.3 g L^?1 total sugars, 68.3 g L^?1 citric acid was produced and the yield of citric acid was 0.91 g g^?1 within 336 h. At the end of the fermentation, 9.2 g L^?1 of residual total sugar and 2.1 g L^?1 of reducing sugar were left in the fermented medium. At the same time, citric acid in the supernatant of the culture was purified. It was found that 67.2 % of the citric acid in the supernatant of the culture was recovered and purity of citric acid in the crystal was 96 %.     

Genetically modified Saccharomyces cerevisiae for one-step fermentation of bioalcohol using corncob as sole carbon source

The corncob is an important biomass for bioalcohol production. However, there is a minor but complicated pretreatment process before it is used for bioalcohol fermentation. In this study, three genetically modified Saccharomyces cerevisiae Y33 strains containing endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BG) genes were constructed. A one-step fermentation process was carried out with the recombinants using corncob as the sole carbon source. In a 3-L fermentation system, the concentration of alcohol reached 2.02 g/L and the concentration of glycerine reached 0.85 g/L after 96 h. The results prove that corncob powder can be utilized effectively by genetically modified Saccharomyces cerevisiae without any chemical pretreatment. The mixed recombinant Saccharomyces cerevisiae cells show effective synergism in the one-step fermentation system. It is feasible that corncob can be used as the sole carbon source in bioalcohol production with a one-step fermentation process.     

A high-throughput method for measuring growth and loss rates in microalgal cultures

A miniaturized and low-cost assay for algal growth and loss rates, and estimation of compensation light was developed and optimized. Microalgal cultures were grown in white 96-well microplates to estimate specific growth rates at six temperatures, five salinities and eight light levels. Data from black 24-well microplates at six temperatures, five salinities and five light conditions were used in addition to estimate loss rates and compensation light. Absorption and reflection of light were different in the white and black microplates. Growth rates were estimated from daily in vivo fluorescence (IVF) measurements using a microplate reader fitted with a fluorometer. To validate the microplate algal growth assay, IVF was compared with cell counting by flow cytometry. Maximal growth rate for the test alga Pseudochattonella farcimen (Heterokonta) was estimated to 0.52?±?0.05 day^?1 at optimal temperatures ranging from 9 to 14°C and salinities 18–26 psu. Lowest value of compensation light as photosynthetic photon flux density (PPFD) was 4.2?±?1.2 μmol photons m^?2 s^?1, and lowest saturation light, 34.1?±?3.7 μmol photons m^?2 s^?1, was observed in the temperature range 5–11°C and salinity range 23–28 psu. Minimum loss rate was obtained at temperatures 5–8°C and salinities 26–31 psu. Blooms of P. farcimen have been recorded in nature under conditions similar to those minimizing loss rates rather than maximizing growth rates in this study. The microalgal assay described here allows for a large number of conditions to be tested, and accurate optimal conditions for growth and loss rates to be obtained.     

Optimization of submerged fermentation conditions for immunosuppressant mycophenolic acid production by Penicillium roqueforti isolated from blue-molded cheeses: enhanced production by ultraviolet and gamma irradiation

Mycophenolic acid (MPA) is a promising drug owing to its immunosuppressive and biological activities. In this study, two strains of Penicillium roqueforti designated as AG101 and LG109 were selected among several strains isolated from Roquefort cheese samples on the basis of their activity for MPA-producing ability. The appropriate fermentation conditions necessary for MPA biosynthesis by the two respective fungal strains were investigated. These conditions included selection of the cultivation medium, agitation rate, incubation temperature, fermentation time, pH value, inoculum size, and fermentation medium volume. Maximum MPA productivities were maintained when the fermentation process was carried out using a medium composed of (g l^?1): Sucrose, 30; peptone, 5.0; KH₂PO₄, 1.0; MgSO₄·7H₂O, 0.5 and KCl, 0.5; pH 6.0, inoculated with an inoculum size of 6.0 % (v/v), and incubated at 25 °C for 10 days at 120 rpm. The potentiality of both P. roqueforti strains for further improvement of MPA production was applied by mutagenesis through exposure to irradiation by ultraviolet rays (UV, 254 nm) for different periods of time and gamma rays at various doses (KGy). The dry cell weight of both irradiated fungal strains showed a greater reduction when irradiated either with UV or gamma rays. However, the MPA yield of both strains was increased by 1.27–1.39 fold when irradiated with UV rays and by 2.11–2.33 fold when irradiated with gamma rays, as compared with the respective controls (non-irradiated cultures). These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

ENGINEERING A HIGH-YIELD GLUTATHIONE STRAIN OF Hansenula polymorpha USING ION BEAM IMPLANTATION

To generate an industrial strain of Hansenula polymorpha capable of yielding greater levels of glutathione (GSH), wild strain H. polymorpha DL-1 cells were mutated using a nitrogen ion beam, a novel mutagen. At an energy level of 20 keV and dose of 2.13 × 10¹⁶ ions/cm², H. polymorpha strain 28 (HP28) with a high-yield of GSH was screened. HP28 intracellular GSH levels reached 337.16 mg/L by ion beam implantation, 1.56 times greater than that of the wild type strain when the fermentation time was shortened from 48 hr to 42 hr, greatly improving efficiency and reducing the cost of industrial-scale production. The enhanced efficiency of HP28 is promising for GSH production from lignocellulosic materials. Therefore, the ion beam implantation would be a cost-effective alternative to the conventional mutation method for engineering yeast and improving its utility.     

Enrichment of biologically active 18-β glycyrrhetinic acid in Glycyrrhiza glabra root by solid state fermentation

This paper describes the in situ bioconversion of glycyrrhizin of Glycyrrhiza glabra root to 18-β glycyrrhetinic acid by solid state fermentation. Fermentation was carried out with two different fungal strains, Penicillium chrysogenum and Rhizopus oryzae. The solid state fermentation was carried out under stationary state and under rotating state. Penicillium chrysogenum is a better producer of 18-β glycyrrhetinic acid than Rhizopus oryzae. The induced P. chrysogenum seed culture produces higher 18-β glycyrrhetinic acid with 2.955 mg g^?1 and maximum β-glucuronidase activity of 3,583.8 U ml^?1 under stationary solid state fermentation. The mycelium growth and bioconversion rate is highest at pH of 5.5 and 4.5, respectively. G. glabra root supplemented with a solution of dextrose 9 g l^?1, MnSO₄?·?H₂O 3 g l^?1 and (NH₄)₂SO₄ 0.540 g l^?1 produces 48.580 mg of 18-β glycyrrhetinic acid per gram of G. glabra root, i.e. 86.74 % bioconversion by P. chrysogenum in 96 h under stationary state solid state fermentation.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.     

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD) gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85 g/L) and yield (0.84 g/g) were achieved by batch fermentation at pH-4.5/30 °C by the YqhD recombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23 g/L and yield of 0.72 g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg²⁺ and Mn²⁺, in TS increased 1,3-PDO yield (0.74 g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.     

Short-term adaptation improves the fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low pH

The release of acetic acid due to deacetylation of the hemicellulose fraction during the treatment of lignocellulosic biomass contributes to the inhibitory character of the generated hydrolysates. In the present study, we identified a strain-independent adaptation protocol consisting of pre-cultivating the strain at pH 5.0 in the presence of at least 4 g L^?1 acetic acid that enabled aerobic growth and improved fermentation performance of Saccharomyces cerevisiae cells at low pH (3.7) and in the presence of inhibitory levels of acetic acid (6 g L^?1). During anaerobic cultivation with adapted cells of strain TMB3500, the specific ethanol production rate was increased, reducing the fermentation time to 48 %.     

EVALUATION OF BIOETHANOL PRODUCTION FROM CAROB PODS BY Zymomonas mobilis AND Saccharomyces cerevisiae IN SOLID SUBMERGED FERMENTATION

Bioethanol production from carob pods has attracted many researchers due to its high sugar content. Both Zymomonas mobilis and Saccharomyces cerevisiae have been used previously for this purpose in submerged and solid-state fermentation. Since extraction of sugars from the carob pod particles is a costly process, solid-state and solid submerged fermentations, which do not require the sugar extraction step, may be economical processes for bioethanol production. The aim of this study is to evaluate the bioethanol production in solid submerged fermentation from carob pods. The maximum ethanol production of 0.42 g g^?1 initial sugar was obtained for Z. mobilis at 30°C, initial pH 5.3, and inoculum size of 5% v/v, 9 g carob powder per 50 mL of culture media, agitation rate 0 rpm, and fermentation time of 40 hr. The maximum ethanol production for S. cerevisiae was 0.40 g g^?1 initial sugar under the same condition. The results obtained in this research are comparable to those of Z. mobilis and S. cerevisiae performance in other culture mediums from various agricultural sources. Accordingly, solid submerged fermentation has a potential to be an economical process for bioethanol production from carob pods.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

Production of butanol and isopropanol with an immobilized <Emphasis Type="Italic">Clostridium</Emphasis>

Clostridium beijerinckii optinoii is a Clostridium species that produces butanol, isopropanol and small amounts of ethanol. This study compared the performances of batch and continuous immobilized cell fermentations, investigating how media flow rates and nutritional modification affected solvent yields and productivity. In 96-h batch cultures, with 80 % of the 30 g L^?1 glucose consumed in synthetic media, solvent concentration was 9.45 g L^?1 with 66.0 % as butanol. In a continuous fermentation using immobilized C. beijerinckii optinoii cells, also with 80 % of 30 g L^?1 glucose utilization, solvent productivity increased to 1.03 g L^?1 h^?1. Solvent concentration reached 12.14 g L^?1 with 63.0 % as butanol. Adjusting the dilution rate from 0.085 to 0.050 h^?1 to allow extended residence time in column was required when glucose concentration in fresh media was increased from 30 to 50 g L^?1. When acetate was used to improve the buffer capacity in media, the solvent concentration reached 12.70 on 50 g L^?1 glucose. This continuous fermentation using immobilized cells showed technical feasibility for solvent production.     

Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.