Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.     

Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor.In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered.Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities.The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.     

Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.     

Overexpression of IbpB enhances production of soluble active Streptomyces olivaceovirdis XynB in Escherichia coli

The small heat shock protein IbpB of Escherichia coli can accelerate protein disaggregation from inclusion body by Hsp100-Hsp70 re-activation system in vitro. It was therefore hypothesized that overexpression of IbpB might be able to promote protein disaggregation from inclusion body, by which more soluble recombinant proteins would be obtained. The overexpression of IbpB actually enhanced production of more active soluble XynB of Streptomyces olivaceovirdis in E. coli BL21(DE3). Surprisingly, the disaggregation of XynB from inclusion body was not accelerated. It seemed that the overexpressed IbpB protected improperly or partially folded XynB from aggregation and mediated the subsequent refolding. These results show potential of improving production of active heterologous proteins in E. coli.     

MHF组蛋白折叠复合体组分编码基因MHF1过表达对重组酿酒酵母纤维素酶生产的影响

【背景】重组酿酒酵母可用于生产多种药用蛋白和工业酶等外源蛋白,但蛋白分泌水平低是限制其异源蛋白高效生产的重要因素。异源蛋白表达和分泌过程可能会对宿主细胞产生多种胁迫,因此,研究胁迫响应相关基因对重组酵母异源蛋白生产的影响具有重要意义。Mhf1p是MHF组蛋白折叠复合体的组分之一,与DNA损伤修复及维持基因组稳定性有关,但其对异源蛋白生产的作用尚不清楚。【目的】研究MHF1过表达对重组酿酒酵母蛋白生产的影响。【方法】在分泌表达纤维素酶的重组酿酒酵母菌株中利用基于CRISPR-Cas9的基因组编辑技术整合过表达MHF1,分析其对产酶的影响,并探讨影响产酶的分子机理。【结果】与出发菌株相比,过表达MHF1菌株的外切纤维素酶CBH酶活性提高了38%。对过表达MHF1的CBH生产菌株中蛋白合成和分泌途径相关基因转录水平进行检测,发现与对照菌株相比,CBH1基因和与分泌相关的SEC22、ERV29等基因在不同时间点呈现不同程度显著上调。【结论】MHF1过表达可促进酿酒酵母异源外切纤维素酶的生产,并影响外源酶基因和分泌途径基因的表达,可能通过对多基因的协同表达影响促进产酶。     

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 ΔackA-pta, ΔpoxB Δpppc ppc-p37 for recombinant β-galactosidase production

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, β-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards β-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.     

Combinatorial network of transcriptional regulation and microRNA regulation in human cancer

Background

In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model??s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes.

Results

We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF) with grouping reaction (GR) constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via ??GR constraints??. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation.

Conclusions

FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm was validated through the experiments on the enhanced production of putrescine in E. coli, in addition to the comparison with the previously reported experimental data. The FVSEOF strategy with GR constraints will be generally useful for developing industrially important microbial strains having enhanced capabilities of producing chemicals of interest.     

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.     

A dynamic,genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression

Recently, lactic acid bacteria (LAB) have attracted a great deal of interest because of their potential to serve as oral delivery vehicles for recombinant protein vaccines. An important limitation to their use is the typically low level of heterologous expression obtained in LAB. To address this, a dynamic flux balance analysis (DFBA) model was used to identify gene targets for increasing specific expression of Green Fluorescent Protein (GFP), a model heterologous protein, in Lactococcus lactis IL1403. Two strains, each targeting one of the top model-identified genes, were constructed and tested in vivo. Data show that both strains, by a conservative estimate, achieved 15% higher GFP per cell than the control strain, a qualitative confirmation of the model predictions. A genome-scale DFBA model for L. lactis growing on M17 medium is presented along with the procedure for screening gene targets and a powerful method for visualizing fluxes in genome-scale metabolic networks.     

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.     

Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches

Pichia pastoris is an efficient expression system for production of recombinant proteins. To understand its physiology for building novel applications it is important to understand and reconstruct its metabolic network. The metabolic reconstruction approach connects genotype with phenotype. Here, we have attempted to reconstruct carbohydrate metabolism pathways responsible for high biomass density and N-glycosylation pathways involved in the post translational modification of proteins of P. pastoris CBS7435. Both these metabolic pathways play a crucial role in heterologous protein production. We report novel, missing and unannotated enzymes involved in the target metabolic pathways. A strong possibility of cellulose and xylose metabolic processes in P. pastoris CBS7435 suggests its use in the area of biofuels. The reconstructed metabolic networks can be used for increased yields and improved product quality, for designing appropriate growth medium, for production of recombinant therapeutics and for making biofuels.     

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.     

Metabolic impact assessment for heterologous protein production in Streptomyces lividans based on genome-scale metabolic network modeling

The metabolic impact exerted on a microorganism due to heterologous protein production is still poorly understood in Streptomyces lividans. In this present paper, based on exometabolomic data, a proposed genome-scale metabolic network model is used to assess this metabolic impact in S. lividans. Constraint-based modeling results obtained in this work revealed that the metabolic impact due to heterologous protein production is widely distributed in the genome of S. lividans, causing both slow substrate assimilation and a shift in active pathways. Exchange fluxes that are critical for model performance have been identified for metabolites of mouse tumor necrosis factor, histidine, valine and lysine, as well as biomass. Our results unravel the interaction of heterologous protein production with intracellular metabolism of S. lividans, thus, a possible basis for further studies in relieving the metabolic burden via metabolic or bioprocess engineering.     

Expanding the landscape of recombinant protein production in Escherichia coli

Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the co-expression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of co-expression to overproduce membrane proteins.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.