植物生物反应器生产异源蛋白

利用植物表达系统生产外源蛋白是一个有吸引力的廉价生产系统 ,它有可能替代外源蛋白的发酵生产系统。本文对分子农业的意义、植物表达外源蛋白的影响因素和植物生产外源蛋白质的研究进展等方面作了论述     

植物渗调蛋白的研究进展

植物在逆境下会产生许多逆境响应蛋白,渗调蛋白（Osmotins)是其中重要的一种,该蛋白是一种逆境适应,蛋白,其基因的表达受到干旱,盐渍,病原侵染,乙烯,ABA等因子的诱导,与植物的抗旱,耐盐和抗病性等有关。作者对渗调蛋白的存在,特性,功能和基因调控规律作以下简要综述。     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

米曲霉异源蛋白表达系统研究进展及展望

米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。     

植物金属硫蛋白研究进展(二)植物MT基因的表达特征及其功能

阐述了植物金属硫蛋白基因表达的组织特异性、金属离子、激素、化学试剂等对植物金属硫蛋白基因表达的影响及植物金属硫蛋白的功能。     

转基因植物表达重组蛋白的研究进展

植物表达系统的一些潜在优点 ,如重组蛋白的高积累水平 ,糖基化 ,细胞内的定位和自然储藏的稳定性是目前植物生产重组蛋白系统研究成为热点的主要原因 .在研究和选择转基因植物表达系统的过程中 ,转化 ,转化后 ,翻译 ,翻译后等环节都会影响到最终产物的数量和质量 ,因此应该了解基因表达的规律 ,以制定植物生产重组蛋白合适的策略 ,重组蛋白积累水平是关键 ,但其它因素如植物的选择 ,转基因植物的处理 ,下游加工等同样重要 .某些情形下 ,仅下游加工的成本一项就影响到特定植物表达系统的实际应用价值 .     

丝状真菌高效表达异源蛋白研究进展

丝状真菌是具有高效分泌蛋白质潜力的真核表达系统, 能对蛋白质进行翻译后修饰, 如蛋白质糖基化等; 并且比植物、昆虫和哺乳动物细胞具有更快的生长速率。近年来, 随着真菌分子遗传技术和菌种改良策略的进步, 尤其是真菌基因组学的发展, 利用丝状真菌生产异源蛋白越来越受到关注。综述了丝状真菌作为细胞工厂生产异源蛋白的最新探索与进展, 其中包括功能基因组学在蛋白表达与分泌研究中的应用, 同时探讨了异源蛋白表达和生产的改进策略。     

转基因植物生产药用蛋白研究进展

转基因植物作为生物反应器能生产医学上有生物活性的药用蛋白,该文对转基因植物生产医药蛋白的种类、途径及优缺点、改进措施进行了阐述。     

异源植物中转座因子标签研究的进展

异源植物中转座因子标签研究的进展瞿绍洪１,２）景健康１）胡含１）（１中国科学院遗传研究所,北京１００１０１;２中国科学院植物研究所,北京１０００９３）ＰｒｏｇｒｅｓｏｆＴｒａｎｓｐｏｓｏｎＴａｇｇｉｎｇｉｎＨｅｔｅｒｏｌｏｇｏｕｓＰｌａｎｔｓＱｕＳｈ...     

丝状真菌高效表达异源蛋白研究进展

丝状真菌是具有高效分泌蛋白质潜力的真核表达系统, 能对蛋白质进行翻译后修饰, 如蛋白质糖基化等; 并且比植物、昆虫和哺乳动物细胞具有更快的生长速率。近年来, 随着真菌分子遗传技术和菌种改良策略的进步, 尤其是真菌基因组学的发展, 利用丝状真菌生产异源蛋白越来越受到关注。综述了丝状真菌作为细胞工厂生产异源蛋白的最新探索与进展, 其中包括功能基因组学在蛋白表达与分泌研究中的应用, 同时探讨了异源蛋白表达和生产的改进策略。     

Evaluation of different promoters for the efficient production of heterologous proteins in baker's yeast

We have constructed reporter gene expression vectors placing the Aspergillus oryzae -amylase cDNA and the Geotrichum candidum lipase 2 gene, under the control of the yeast gene promoters,ACT1, ADH1, PGK1, and TDH1. Expression regulated by the shorted form of the ADH1 promoter gave the highest proteins yield for cells cultured in molasses medium or flour/water mixtures. Nevertheless, the ACT1 promoter appeared as the strongest in cells growing actively with sucrose.     

普鲁兰酶的异源表达、结构解析及分子改造研究进展

淀粉是由葡萄糖单元通过α-1,4-葡萄糖苷键和α-1,6-葡萄糖苷键连接而成,不仅是食物的主要成分,也是淀粉深加工工业的基本原料来源。普鲁兰酶能够高效水解淀粉分子中的α-1,6-葡萄糖苷键,与其他的淀粉加工酶复合使用,能够有效提高淀粉的利用率,在淀粉深加工工业中具有“提质增效”的重要作用。本文综述了普鲁兰酶产酶菌株的筛选及编码基因的克隆表达,总结了表达元件及发酵条件优化对普鲁兰酶产酶水平的影响,探讨了普鲁兰酶结构解析及分子改造等方面的研究进展。同时分析了当前研究中存在的问题,并对未来的研究进行了展望,以期为普鲁兰酶的研究及应用提供参考和启示。     

Yeast systems biotechnology for the production of heterologous proteins

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering.     

Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.     

Strategies for improving heterologous protein production from filamentous fungi

Despite the naturally high capacity for protein secretion by many species of filamentous fungi, secteted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains and screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others.Approaches used in heterologous protein production from filamentous fungi are discussed and a perspective on emerging strategies is presented.     

Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.     

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

Improved heterologous erythromycin A production through expression plasmid re‐design

The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli‐derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species‐specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5‐fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:862–869, 2013