毕赤酵母外源蛋白分泌途径的研究进展

毕赤酵母作为一种重要的表达外源蛋白的宿主,提高其外源蛋白的分泌量非常有必要。近年来很多学者报道了与毕赤酵母外源蛋白分泌相关的基因、蛋白质,同时毕赤酵母基因组的公布加快了这方面的研究进展。文章根据外源蛋白分泌的途径,分步骤地总结了涉及的基因和蛋白,有利于分析控制蛋白分泌效率的具体步骤,为构建更加高效的毕赤酵母表达系统提供参考。     

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115

Background

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD⁺ or NADP⁺ as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet.

High-level expression of biologically active glycoprotein hormones in <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated <Superscript>15</Superscript>N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active ¹⁵N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH₄Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man_8–15GlcNAc₂). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Man_up-to-30GlcNAc₂). The latter finding made the X-33 strain not very suitable for generating ¹⁵N-labeled material. Therefore, ¹⁵N-phCG was expressed in the GS115 strain using the new optimized protocol. The ¹⁵N-enrichment was evaluated by ¹⁵N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that ¹⁵N-phCG/GS115 had the same folding as urinary hCG. Furthermore, ¹⁵N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays. Véronique Blanchard and Rupali A. Gadkari contributed equally.     

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris)

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and ‘greener’ food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with β-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has β-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

     

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β-galactosidase under FLD promoter, have been constructed.     

Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum ω-transaminase alone or combined with native transketolase

Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration or robustness to fluctuating pH, osmolarity and biocontaminant load in feedstocks. The methylotrophic yeast Komagataella phaffii is an attractive alternative to Escherichia coli for whole cell biocatalysis due to its genetic tractability and capacity to grow to up to 60% wet cell weight by volume. We sought to exploit high cell density K. phaffii to intensify whole-cell chiral amino-alcohol (CAA) biosynthesis. We engineered two novel K. phaffii GS115 strains: one by inserting a Chromobacterium violaceum ω-transaminase CV2025 transgene, for strain PpTAmCV708, and a second strain, PpTAm-TK16, by also inserting the same CV2025 transgene plus a second transgene for a native transketolase. At high cell density, both strains tolerated high substrate concentrations. When fed three low cost substrates, 200 mM glycolaldehyde, 200 mM hydroxypyruvate and 150 mM methylbenzylamine, PpTAm-TK16 whole cells achieved 0.29 g L⁻¹ hr⁻¹ space–time yield of the acetophenone by-product, a 49-fold increase of the highest levels reported for E. coli whole cells harboring the equivalent pathway. When fed only the low-cost substrate, 150 mM methylbenzylamine, strain PpTAmCV708 achieved a 105-fold increase of reported E. coli whole cell biocatalysis performance, with a space–time yield of 0.62 g L⁻¹ hr⁻¹ of the CAA, 2-amino-1,3,4-butanetriol (ABT). The rapid growth and high biomass characteristics of K. phaffii were successfully exploited for production of ABT by whole-cell biocatalysis at higher levels than the previously achieved with E. coli in the presence of the same substrates.     

Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase

The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5–7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression.     

产肌醇毕赤酵母细胞工厂的优化

【背景】肌醇是一种B族维生素,广泛应用于食品、医药、饲料等领域。微生物发酵法是最具前景的肌醇生产方法,但使用大肠杆菌生产的肌醇在食品及医药领域中的使用受到限制。毕赤酵母作为生物安全菌株是工业上生产异源蛋白的良好宿主,其本身含有天然的肌醇合成途径,具有被改造成为高效生产肌醇细胞工厂的潜力。【目的】通过代谢工程改造毕赤酵母工程菌株,降低副产物的生成并提高肌醇的产量。【方法】以实验室前期构建的产肌醇毕赤酵母工程菌株为出发菌株,确定副产物阿拉伯糖醇、核糖醇和甘露糖合成相关基因。通过关键基因敲除、发酵液中葡萄糖浓度控制降低副产物的产量。通过过表达甘油转运蛋白、甘油激酶和甘油-3-磷酸脱氢酶基因实现产肌醇毕赤酵母对甘油和葡萄糖的共利用,得到重组菌Z10。经过发酵条件优化,进一步提高Z10的肌醇产量。【结果】在最优条件下,重组菌Z10的肌醇产量达到36.7 g/L,是目前酵母类细胞工厂生产肌醇的最高值,副产物总产量与出发菌株相比降低了63.1%。【结论】在毕赤酵母中建立了降低阿拉伯糖醇、核糖醇和甘露糖合成的有效策略,并通过甘油、葡萄糖共利用及相对应的发酵条件优化提高了肌醇产量,为肌醇及其他高价值生物...     

巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析

【目的】基于转录组学技术研究表达磷脂酶A_2的毕赤酵母重组菌在甲醇诱导表达外源蛋白时的基因表达差异,从而解析外源蛋白高效诱导表达机制,为进一步工程菌株的改造提供理论支撑。【方法】以一株产磷脂酶(PLA_2)的毕赤酵母为出发菌株,采用RNA-Seq二代测序方法,研究在甘油培养和甲醇诱导两种条件下,重组毕赤酵母转录组基因表达差异情况。【结果】重组毕赤酵母中共鉴定到5225个转录本。甘油培养与甲醇诱导相比,共有857个基因发生显著变化。依据代谢途径分类,差异基因集中在核糖体成分、甲醇代谢、磷酸戊糖途径、糖酵解途径、柠檬酸循环、乙醛酸循环以及蛋白质加工过程。【结论】通过分析甲醇诱导前后的差异表达基因,结果表明碳源改变对胞内代谢会产生全局影响。本研究结果为进一步研究毕赤酵母表达外源蛋白的机制提供了基础。     

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_n (n?=?2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96?hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.     

Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

Genotyping of recombinant Pichia pastoris strains

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.     

Titer improvement of iso-migrastatin in selected heterologous <Emphasis Type="Italic">Streptomyces</Emphasis> hosts and related analysis of mRNA expression by quantitative RT–PCR

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO₃ to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.     

Cloning and Expression of Pseudomonas taetrolens Y-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.     

鲈鱼生长激素在甲醇酵母中的胞内表达

甲醇酵母pichia pastoris是一种理想的真核蛋白高水平表达系统.将鲈鱼(Lateolabrax japonicus)生长激素基因克隆到酵母整合型质粒载体pHIL-D2,经转化his4缺陷型酵母GS115,用PCR方法筛选阳性转化子,并用斑点印迹法筛选多拷贝转化子,经甲醇诱导表达,SDS-PAGE和蛋白质印迹杂交结果证实了表达产物为重组的鲈鱼生长激素.     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Improvement of the efficiency and quality in developing a new CHO host cell line

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.     

Influence of different host associations on glutamine synthetase activity and ammonium transporter in <Emphasis Type="Italic">Santalum</Emphasis><Emphasis Type="Italic">album</Emphasis> L.

The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica—sandal association compared to other leguminous and non-leguminous host associations. The association of N₂ fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the \( 2^{{ - \Delta \Delta {\text{C}}_{\text{T}} }} \) method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.     

Protoplast transformation of Kluyveromyces marxianus

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 °C with carrier DNA, CaCl₂, and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3–4% agar and 0.8 m sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8 × 10⁴ transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.