Optimization of agitation and aeration conditions for maximum virginiamycin production

To maximize the productivity of virginiamycin, which is a commercially important antibiotic as an animal feed additive, an empirical approach was employed in the batch culture of Streptomyces virginiae. Here, the effects of dissolved oxygen (DO) concentration and agitation speed on the maximum cell concentration at the production phase, as well as on the productivity of virginiamycin, were investigated. To maintain the DO concentration in the fermentor at a certain level, either the agitation speed or the inlet oxygen concentration of the supply gas was manipulated. It was found that increasing the agitation speed had a positive effect on the antibiotic productivity independent of the DO concentration. The optimum DO concentration, agitation speed and addition of an autoregulator, virginiae butanolide C (VB-C), were determined to maximize virginiamycin productivity. The optimal strategy was to start the cultivation at 450 rpm and to continue until the DO concentration reached 80%. After reaching 80%, the DO concentration was maintained at this level by changing the agitation speed, up to a maximum of 800 rpm. The addition of an optimal amount of the autoregulator VB-C in an experiment resulted in the maximal production of virginiamycin M (399 mg/l), which was about 1.8-fold those obtained previously. Received: 13 July 1998 / Received revision: 19 August 1998 / Accepted: 13 September 1998     

Optimum autoregulator addition strategy for maximum virginiamycin production in batch culture of Streptomyces virginiae

Virginiae butanolides (VBs) are autoregulators of Streptomyces virginiae, which induce virginiamycin biosynthesis. Generally, autoregulators are synthesized by the microorganism itself during culture. Addition of chemically synthesized virginiae butanolide-C (VB-C), which is one of the VBs, can also control the induction time and the amount of virginiamycin production. The optimum concentration and shot-feeding time of VB-C for the maximum production of virginiamycins M and S were investigated in flasks and jar-fermentor batch cultures. VB-C addition later than 8 h from the start of culture induced not only virginiamycin M and S synthesis but also VB synthesis. Virginiamycin M and S production increased with the decrease of total VBs (produced VBs and added VB-C) concentration. That is, although VBs are needed to induce virginiamycin M and S synthesis, the amount of VB-C added should be such that as small an amount as possible of VBs is synthesized to achieve the maximum production of virginiamycins M and S. However, the VB-C addition earlier than 8 h from the start of culture showed no clear relationship between the amounts of VBs and virginiamycins M and S produced. In conclusion, the maximum production of virginiamycins M and S was attained by the shot addition of 5 mug/L VB-C at 8 h from the start of culture. The maximum value was about twofold that without VB-C addition. The optimum addition strategy of VB-C was confirmed by the jar-fermentor experiments. (c) 1995 John Wiley & Sons, Inc.     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

barS1, a gene for biosynthesis of a gamma-butyrolactone autoregulator,a microbial signaling molecule eliciting antibiotic production in Streptomyces species

From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M(1) and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the gamma-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M(r), 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to beta-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the DeltabarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the DeltabarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.     

Identification by gene deletion analysis of <Emphasis Type="Italic">barS2</Emphasis>, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in <Emphasis Type="Italic">Streptomyces virginiae</Emphasis>

Virginiae butanolide (VB) is a member of the gamma-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M1 and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW2 located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae.     

The nusG gene of Streptomyces griseus: Cloning of the gene and analysis of the A-factor binding properties of the gene product

Abstract The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identify of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae . NusG protein was expressed in Escherichia coli . However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.     

IM2, a new inducer of blue pigment production in Streptomyces sp. MAFF 10-06015

A new autoregulator designated as IM2, which induces blue pigment production in Streptomyces sp. MAFF 10-06015, was discovered. The culture conditions developed here for the production of the pigment by the strain did not require the addition of an artificial inducer such as γ-nonalactone or the autoregulator of S. virginiae MAFF 10-06014, IM, which induces the production of virginiamycin by this microorganism. The major improvements in the culture conditions for spontaneous pigment production included the inoculation conditions and the dilution of the medium. The method of IM2 assay was established and the time courses of IM2 production were followed in the cultures using flasks and a jar fermentor. It was confirmed that IM2 released once into the culture filtrate from the cells was taken up into the cells again. The concentration of IM required to induce pigment production in Streptomyces sp. MAFF 10-06015 was 50 u·ml⁻¹. However a concentration of 200 u·ml⁻¹ of IM2 was unable to induce the production of virginiamycin in S. virginiae MAFF 10-06014.     

Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.     

Purification and characterization of the IM-2-binding protein from Streptomyces sp. strain FRI-5.

IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-(hydroxymethyl)butanolide] of Streptomyces sp. strain FRI-5 is one of the butyrolactone autoregulators of Streptomyces species and triggers production of blue pigment as well as the nucleoside antibiotics showdomycin and minimycin. A tritium-labeled IM-2 analogue, 2,3-trans-2(1'-beta-hydroxy-[4',5'-3H]pentyl)-3-(hydroxymethyl)butano lide ([3H]IM-2-C5; 40 Ci/mmol), was synthesized for a competitive binding assay, and an IM-2-specific binding protein was found to be present in the crude cell extract of Streptomyces sp. strain FRI-5. During cultivation for 24 h, the specific IM-2-binding activity increased rapidly, reached a plateau at 10 to 14 h, and declined sharply thereafter, showing only 6% activity after 24 h of cultivation. A Scatchard plot of the binding data demonstrated that the dissociation constant (Kd) for [3H]IM-2-C5 was 1.3 nM, while the Kd for a 3H-labeled virginiae butanolide (VB) analogue, 2-(1'-alpha-hydroxy-[6',7'-3H]heptyl)-3-(hydroxymethyl)butanolide ([3H]VB-C7), another butyrolactone autoregulator possessing the opposite configuration at C-1' was 35 nM. Furthermore, at a 15-fold molar excess, the effectiveness of several autoregulators as nonlabeled competitive ligands against [3H]IM-2-C5 was IM-2 type > VB-C type >> A-factor type, indicating that the binding protein in Streptomyces sp. strain FRI-5 is highly specific toward IM-2. Ultracentrifugation showed that the IM-2-binding protein is present almost exclusively in the 100,000 x g supernatant fraction, indicating that the binding protein is a cytoplasmic soluble protein. The binding protein was purified by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-100 HR gel filtration, DEAE-5PW high-performance liquid chromatography (HPLC), and phenyl-5PW HPLC. The apparent Mr of the native IM-2-binding protein as determined by molecular sieve HPLC was about 60,000 in the presence of 0.5, 0.3, or 0.1 M KCl, while by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was about 27,000, suggesting that the native binding protein is present in the form of a dimer.     

Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in Streptomyces virginiae

Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M₁ and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW ₂ located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae. An erratum to this article can be found at     

重组巴氏毕赤酵母高密度发酵表达rHSA

对基因工程菌Pichiapastoris的摇瓶发酵条件进行了试验 ,并根据摇瓶发酵的优化结果进行了补料分批高密度发酵。在摇瓶发酵时 ,甲醇诱导基因工程菌P .pastoris表达重组人血清白蛋白的发酵周期为 96h ;甲醇的最佳诱导浓度为 1 0g L ;发酵pH范围为 5 72～ 6 5 9;在摇瓶培养时 ,随着接种量的增加 ,虽然目的蛋白表达量缓慢增加 ,但单位细胞光密度的蛋白产率却明显下降 ,符合y =1 2 941x- 0 50 59方程 (线性相关系数r=0 9789) ,其限制性因子很可能为溶氧。在分批发酵 ,接种量为 1 0 %且种子细胞光密度 (OD60 0 )为 2 0左右时 ,细胞生长的延迟期为 2 1 1h左右 ,细胞生长光密度与培养时间的关系模型为 :y =0 7841e0 .2 3 19t(线性相关系数r=0 .993 6 ) ;在补料发酵时细胞干重浓度可达到 1 1 5g L— 1 6 0g L ,在 1 2 0h重组人血清白蛋白表达量最大达到 3 6g L。     

Phenotypic and genotypic characterization of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae

A representative set of 19 mutants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all mutants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter.     

Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub

The bacterium Serratia entomophila (Enterobacteriaceae) has been developed as a commercially available biopesticide for control of the pasture pest Costelytra zealandica. The influence of culture medium composition, dissolved oxygen (DO) concentration and harvesting time were investigated in order to optimise the production of S. entomophila. In batch fermentations, highest yields were achieved using sucrose (40 g L^-1) as the carbon source, followed closely by fructose and molasses. The effect of yeast extract (YE), marmite and bakery yeast as cell growth enhancers was also examined in both batch and fed-batch mode. Culture medium containing 20 g L^-1 of YE (fed-batch) produced the highest cell density. No significant effect on cell yield was detected when cultures were supplemented with bakery yeast or marmite. The DO concentration influenced biomass production: a 5-fold increase in cell density was achieved when the concentration of DO was maintained in the range of 20-50% (5.7×10¹⁰ CFUs mL^-1) in comparison with 1% (1.2×10¹⁰ CFUs mL^-1). In cultures maintained at 1 and 20% DO concentration, cells harvested from the exponential growth phase survived for less than 2 weeks when stored at 4°C. In contrast, high cell survival (85-100%) was achieved when cells were harvested after they had entered the stationary growth phase. Recommendations are provided for the production of robust, high cell density cultures of S. entomophila.     

葡萄糖流加策略对基因工程FR-008／杀念菌素衍生物CS103补料分批发酵过程的影响

以组合生物合成技术得到的链霉菌FR-008突变株CS103为研究对象,研究了3．7L发酵罐上维持一定葡萄糖浓度对其次级代谢产物脱羧FR-008／candicidin衍生聚酮抗生素CS103生物合成的影响。当初始葡萄糖浓度20g／L,发酵过程还原糖浓度维持在10g/L时,抗生素CS103最高产量较分批发酵最高产量相比提高30％。研究了3．7L罐上补料分批发酵生产CS103的工艺,主要考察了脉冲补料、间歇流加补料和连续流加补料三种补料分批发酵工艺,并与分批发酵进行了比较。连续流加补料维持糖浓度的效果明显,最高产量达到126．9μg/mL,与分批发酵相比提高了44％左右。     

Improved production of viable cells of the salt-tolerant yeast Zygosaccharomyces rouxii by continuous culture

Summary A continuous culture system of the salt-tolerant yeast Zygosaccharomyces rouxii (soy yeast) was investigated in order to obtain high production efficiency of viable cells. The optimum pH and C/N ratio of the feed medium for cell production were about 5.0 and 16–20, respectively. About a fivefold increase in viable cell number and cell productivity (viable cell number per litre per hour) were obtained in glucose-limited culture at a dilution rate (D) of 0.06 h^–1 as compared with batch culture. However, the fermentative activity of the cells from glucose-limited culture was significantly lower than those from batch and dissolved-oxygen (DO)-limited cultures, and the former cells showed lower specific activity of glycolytic enzymes. On the other hand, at the boundary conditions between glucose and DO limitation almost the same cell productivity and higher fermentative activity of the cell were obtained as compared with glucose-limited conditions. The cultivation continued for about 60 days without any problems even if the D was altered. It was found that the continuous cultivation method was suitable for industrial production of viable cells of soy yeasts. Offprint requests to: T. Hamada