Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host, Aspergillus awamori var. kawachi.

A modified glucoamylase gene (glaA) with an extra Thr- and Ser-rich Gp-I domain (T. Semimaru, M. Goto, K. Furukawa, and S. Hayashida, Appl. Environ. Microbiol. 61:2885-2890, 1995) was introduced into a mutant parental host, Aspergillus awamori var. kawachi, in which the original glaA gene had been completely deleted and replaced with the hygromycin phosphotransferase gene. The modified glaA was successfully expressed and secreted. The modified glucoamylase possessed higher digestibility of raw corn starch and higher stabilities in response to heat and extreme pH.     

Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi.

Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that cleavage of the precursor enzyme by autocatalysis caused secretion of multiple amylases by Aspergillus niger

The observation that a mutant strain of Aspergillus niger isolated for protease overproduction accumulated Taka-amylase supported an earlier report that processing of the precursor amylase by protease resulted in the secretion of multiple amylases. Studies using a mutant strain revealed that such processing was not due to aspergillopepsin but to autocatalysis by an inherent protease activity of the precursor and glucoamylase. Alignment of protease sequences with glucoamylase showed regions of consensus with serine carboxypeptidase of A. niger. Thus point mutations in this region due to ultraviolet radiation apparently caused the mutant to evolve with enhanced protease activity that degraded the precursor and accumulated Taka-amylase.     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis

The optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels.     

黑曲霉糖化酶在酿酒酵母中的表达和分泌

从黑曲霉糖化酶高产株T2l合成的糖化酶cDNA,经5’端和3’端改造后克隆到酵母质粒YFDl8上,转化酿酒酵母。转化子的淀粉培养基平板检测,培养滤液蛋白电泳和糖化酶活力分析都表明,含有糖化酶基因表达质粒的酵母转化子能有效地分泌有功能的糖化酶到细胞外。实验证明酵母a园子启动子和分泌信号序列能促使黑曲霉糖化酶cDNA在酵母中表达和分泌．实验还表明．黑曲霉糖化酶原的翻译后加工序列很可能亦能被酵母识别,加工生成有功能的成熟的糖化酶。以上成功为构建有实用意义的淀粉水解酵母工程菌迈出了重要的一步。     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Production of raw cassava starch-digestive glucoamylase by a 2-deoxyglucose-resistant mutant of Rhizopus sp.

In order to improve the productivity of raw cassava starch-digestive glucoamylase of Rhizopus sp. MB46 in a liquid culture, a mutant strain, AF-1, which is resistant to 2-deoxyglucose, was derived. The mutant strain produced glucoamylase in the presence of 0.5% glucose though the parent strain did not. With a rice bran liquid medium the productivity was over 2-times that of the wild type strain. A rice bran liquid medium supplemented with β-cyclodextrin was also effective for glucoamylase production. Other maceration enzymes were also produced at a higher level with mutant strain AF-1 than with the wild type strain in a liquid culture as well as in a solid culture. The elution patterns of these enzymes on CM-cellulose column chromatography were principally the same with both strains except for glucoamylase. When 10% of raw cassava starch and cassava waste were digested with the culture filtrate of mutant strain AF-1, glucose was produced in 7% after 60-h incubation and 3.2% after 48-h incubation, respectively.     

Specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.

alpha-, beta-, and gamma-cyclodextrins (CDs) completely inhibited raw starch digestion by glucoamylase I (GA I, MW 90,000) from Aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of GA I at the CD concentrations of 1-5 mM. CDs at 1-5 mM did not inhibit gelatinized starch hydrolysis by GA I, but at the concentration of 50 mM, they inhibited such hydrolysis slightly. GA I was specifically adsorbed onto CD-Sepharose 6B, but glucoamylase I' (GA I', MW 73,000), which does not adsorb onto or digest raw starch, from the same strain was not adsorbed onto that gel. The adsorption of the glucoamylases onto raw starch and CD-Sepharose 6B was correlated to their digestion of raw starch. The hydrophobic adsorption of GA I onto CDs and raw starch occurred competitively at the Cp region, which is on the C-terminal side of Gp-I in the site for raw starch affinity of GA I, and inclusion complexes were formed.     

Use of catabolic repression in the selection of the glucoamylase-producer Aspergillus niger

The effect of various carbon sources and cAMP on the glucoamylase synthesis in Aspergillus niger was studied to find carbon sources repressed the enzyme synthesis and conditions for the selection of catabolite stable mutants. Maltose at a concentration of 0.5% stimulated the glucoamylase synthesis, but at a concentration of 4% it repressed not only the enzyme synthesis but the growth of the parental strain on the agar medium. The more active mutant 66 was obtained as a result of treatment of Asp. niger st 6 with NG. This mutant is able to grow on the Czapek's medium containing maltose at concentrations 4 or 6%. The mutant 66 produced about 2.9 times more glucoamylase than its parent when maltose was added at 0.5% concentration to the medium. The glucoamylase synthesis in the parental strain was completely repressed under repressing conditions, while the level of the mutant strain activity was 35% from the level of enzyme activity on the medium without the repressor. The addition of cAMP (5.10(-5] resulted in a partial release of maltose (4%) repression of the glucoamylase synthesis in both strains. The results obtained indicate a possibility to select Asp niger mutants with the partially derepressed glucoamylase synthesis. Other regulation mechanisms in addition to catabolite repression may be involved in the regulation of the glucoamylase synthesis.     

基于比速率及代谢流的黑曲霉突变株和野生株分析

黑曲霉具备优异的外源蛋白表达和分泌能力,从而被广泛应用于工业酶制剂的生产。通过研究黑曲霉突变株和野生株在相同培养条件下生理参数和代谢流的差异,确定了黑曲霉合成糖化酶过程中的限制性因素。宏观动力学分析发现,较之野生株,突变株具有较高的最大比生长速率,并且副产物得率降低了90%,底物利用率提高了近30%,表明突变株与野生株在碳源分配和产物转化率上具有明显的差异。利用流平衡分析（FBA）计算胞内代谢通量分布,发现还原力和核糖的供应水平是限制菌体合成的主要因素,而前体氨基酸是合成糖化酶最主要的限制性因素。这些研究结果为后续发酵工艺优化和菌株基因改造提供了有益的思路。     

Combined use of growth rate correlated and growth rate independent promoters for recombinant glucoamylase production in Fusarium venenatum

Fusarium venenatum JeRS 325, a strain which produces recombinant glucoamylase under control of a growth rate independent promoter was transformed with a plasmid carrying the Aspergillus niger glucoamylase gene under control of its own growth rate correlated promoter. Some disruption of the original recombinant genes occurred and at pH 5.8 the double transformant did not produce as much glucoamylase as JeRS 325 in batch culture. However, the double transformant still produced as much glucoamylase as JeRS 325 in fed-batch cultures, illustrating the potential for the combined use of growth rate independent and dependent promoters to improve production of recombinant proteins in fed-batch culture systems.     

黑曲霉糖化酶cDNA的改造及其在酿酒酵母中的表达

应用PCR技术扩增黑曲霉糖化酶cDNA不含非编码区50bp的5’端740bp的序列与该cDNA3’端1400bp的序列连接,获得切除了5’端非编码的糖化酶cDNA。将改造后的cDNA插到质粒pMA91的酵母PGK基因的启动子和转录终止信号之间,构建了含黑曲霉糖化酶基因的表达载体pMAG17。用原生质体转化法将重组质粒pMAG17引入酿酒酵母GRF18。酿酒酵母GRF18转化子在淀粉平板上产生水解透明圈,表明糖化酶已在酵母中表达并分泌至培养基中。测定转化子的胞外酶活力及淀粉水解率。结果表明：改造后的糖化酶基     

Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.     

Expression and secretion of barley α-amylase and A.niger glucoamylase inSaccharomyces cerevisiae

cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol.     

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.     

Production of Hormoconis resinae glucoamylase P by a stable industrial strain of Saccharomyces cerevisiae

A stable strain of Saccharomyces cerevisiae secreting glucoamylase (EC 3.2.1.3) with high debranching activity was constructed using recombinant DNA technology. An expression cassette without bacterial sequences, containing Hormoconis resinae glucoamylase P cDNA and the dominant selection marker MEL1 was integrated into the yeast chromosome using ARS1 homology. The glucoamylase expression level of the integrant yeast strain was increased by chemical mutagenesis. The yeast strains secreting glucoamylase were able to grow on soluble starch (5%, w/v) and ferment it to ethanol.Correspondence to: A. Vainio     

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol. Project supported by the Guangdong Natural Science Foundation.