Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

木质纤维素降解酶生产菌株的遗传改造及应用

通过纤维素酶将木质纤维素向生物新能源的转化对经济社会的可持续发展具有重要意义,被用于纤维素酶制剂工业化生产的微生物大多属于丝状真菌,但丝状真菌的遗传操作困难,且纤维素酶诱导机制尚未阐明,严重制约了纤维素酶高产菌株选育与应用。本文综述了近年来纤维素酶高产菌株遗传操作方法的进展,重点论述了丝状真菌合成纤维素酶过程中的信号感应、信号传导、转录调控的研究,通过理性改造以提高纤维素酶生产菌株的产酶能力,并且总结展望了丝状真菌在工业生产中的应用。     

Members of the Penicillium chrysogenum Velvet Complex Play Functionally Opposing Roles in the Regulation of Penicillin Biosynthesis and Conidiation

A velvet multisubunit complex was recently detected in the filamentous fungus Penicillium chrysogenum, the major industrial producer of the β-lactam antibiotic penicillin. Core components of this complex are P. chrysogenum VelA (PcVelA) and PcLaeA, which regulate secondary metabolite production, hyphal morphology, conidiation, and pellet formation. Here we describe the characterization of PcVelB, PcVelC, and PcVosA as novel subunits of this velvet complex. Using yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), we demonstrate that all velvet proteins are part of an interaction network. Functional analyses using single- and double-knockout strains clearly indicate that velvet subunits have opposing roles in the regulation of penicillin biosynthesis and light-dependent conidiation. PcVelC, together with PcVelA and PcLaeA, activates penicillin biosynthesis, while PcVelB represses this process. In contrast, PcVelB and PcVosA promote conidiation, while PcVelC has an inhibitory effect. Our genetic analyses further show that light-dependent spore formation depends not only on PcVelA but also on PcVelB and PcVosA. The results provided here contribute to our fundamental understanding of the function of velvet subunits as part of a regulatory network mediating signals responsible for morphology and secondary metabolism and will be instrumental in generating mutants with newly derived properties that are relevant to strain improvement programs.     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.     

Role of cultivation media in the development of yeast strains for large scale industrial use

The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.     

Characterization of the α-mannosidase gene family in filamentous fungi: N-glycan remodelling for the development of eukaryotic expression systems

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the α-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.     

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum

Current industrial production of beta-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of beta-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.     

Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples

The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine. We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria. A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes. By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol. Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp. A3, Streptomyces sp. A5 and Streptomyces sp. A13, were able to accumulate this compound in both liquid media and cultures over cork. These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.     

Expression and export: recombinant protein production systems for Aspergillus

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.     

丝状真菌中纤维素酶与半纤维素酶的合成调控简

综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现：细胞外信号分子（C源、光信号）,转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。     

嗜热毁丝霉高通量培养技术及其应用

【背景】丝状真菌是一类重要的工业发酵生产宿主菌,如何进行高通量纯菌培养和高效检测筛选性能优异的菌株是工业丝状真菌研究的重要方向。【目的】研究建立丝状真菌的高通量培养技术并测试应用效果。【方法】通过对丝状真菌培养过程中的制种、接种、培养和检测研究,建立基于孔板的高通量培养技术,并以嗜热毁丝霉为例对该技术进行验证。【结果】与传统的平板制种和摇瓶接种培养方式相比,高通量孔板的培养方式将制种通量提高24倍,单位面积产孢子能力提高350%,液体培养转接效率提高10-40倍,并建立96孔板测定乙醇含量的高通量检测技术。【结论】将丝状真菌的培养和检测通量提高1-2个数量级,为快速检测丝状真菌改造过程产生的大量性状不同菌株并获得目标菌株奠定基础,为丝状真菌高通量筛选研究提供应用指导价值。     

Escape from Premature Death Due to Nuclear Mutations in Podospora anserina: Repeal versus Respite

Premature death has been defined as a growth stoppage linked to the accumulation of specific deletions of the mitochondrial genome (mtDNA) in Podospora anserina. This occurs only in strains carrying the AS1-4 mutation which lies in a gene encoding a cytosolic ribosomal protein. Here we describe the isolation and genetic characterization of 10 nuclear mutations which either delay the appearance of this syndrome (respite from premature death) or cause a switch to the classical senescence process (repeal of premature death). These mutations lie in at least six genes. Some cause defects at the levels of ascospore germination, growth rates, and/or sensitivity toward inhibitors of protein syntheses. All modify the onset of senescence in wild-type (AS1+) strains. The role played by these genes is discussed with respect to the control of diseases due to mtDNA rearrangements in filamentous fungi. Copyright 1998 Academic Press.     

Penicillium chrysogenum Takes up the Penicillin G Precursor Phenylacetic Acid by Passive Diffusion

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.     

Management of palm oil mill effluent through production of cellulases by filamentous fungi

A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to evaluate the bioconversion process.     

New tools for the genetic manipulation of filamentous fungi

Filamentous fungi have a long-standing tradition as industrial producers of primary and secondary metabolites. Initially, industrial scientists selected production strains from natural isolates that fulfilled both microbiological and technical requirements for economical production processes. Subsequently, genetically modified strains with novel properties were obtained through traditional strain improvement programs relying mostly on random mutagenesis. In recent years, however, recombinant technologies have contributed significantly to improve the capacities of production and have also allowed the design of genetically manipulated strains. These major advances were only made possible by basic research bringing deeper and novel insights into cellular and molecular fungal processes, thus allowing the design of genetically manipulated strains. This better understanding of fundamental genetic processes in model organisms has resulted in the design and generation of new experimental transformation strategies to manipulate specifically gene expression and function in diverse filamentous fungi, including those having a biotechnical significance. In this review, we summarize recent developments in the application of homologous DNA recombination and RNA interference to manipulate fungal recipients for further improvement of physiology and development in regards to biotechnical and pharmaceutical applications.     

Physiological description of multivariate interdependencies between process parameters,morphology and physiology during fed‐batch penicillin production

Optimization of productivity and economics of industrial bioprocesses requires characterization of interdependencies between process parameters and process performance. In the case of penicillin production, as in other processes, process performance is often closely interlinked with the physiology and morphology of the organism used for production. This study presents a systematic approach to efficiently characterize the physiological effects of multivariate interdependencies between bioprocess design parameters (spore inoculum concentration, pO₂ control level and substrate feed rate), morphology, and physiology. Method development and application was performed using the industrial model process of penicillin production. Applying traditional, statistical bioprocess analysis, multivariate correlations of raw bioprocess design parameters (high spore inoculum concentration, low pO₂ control as well as reduced glucose feeding) and pellet morphology were identified. A major drawback of raw design parameter correlation models; however, is the lack of transferability across different process scales and regimes. In this context, morphological and physiological bioprocess modeling based on scalable physiological parameters is introduced. In this study, raw parameter effects on pellet morphology were efficiently summarized by the physiological parameter of the biomass yield per substrate. Finally, for the first time to our knowledge, the specific growth rate per spore was described as time‐independent determinant for switching from pellet to disperse growth during penicillin production and thus introduced as a novel, scalable key process parameter for pellet morphology and process performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:689–699, 2014     

Genome sequence of Acremonium strictum AAJ6 strain isolated from the Cerrado biome in Brazil and CAZymes expression in thermotolerant industrial yeast for ethanol production

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.