Heterologous Protein Synthesis Research Articles

Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains.

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate MutS strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii MutS strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.

Plasmids for Efficient Production of Recombinant Proteins in E. coli

Plasmids play a crucial role in the transmission of genetic information across different types of bacteria. Plasmids transfer pathogenicity and survival genes to the host bacterium, allowing the bacteria to adapt to new environments and grow. Several plasmids of differing sizes have been recovered from numerous bacterial species. Plasmids may be used to genetically modify bacteria for a variety of reasons, including the generation of recombinant proteins. Escherichia coli is the most often utilised bacterium for the production of recombinant proteins owing to its quick growth rate, low cost, high yield of recombinant proteins, and simple scale-up procedure. To boost the synthesis of heterologous proteins in E. coli, a number of plasmids have been used. Diverse plasmids have been devised and built to address challenges such as protein refolding, E. coli codon use, a lack of post-translational modifications such as glycosylation, and insufficient recovery of functionally viable recombinant proteins. Recent technical advancements that have made it possible for the E. coli expression system to create more complex proteins, such as glycosylated recombinant proteins and therapeutic antibodies.

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

Aspergillus spp., a versatile cell factory for enzymes and metabolites: Interventions through genome editing

Aspergillus sp. is widely distributed in nature and plays significant roles in the degradation of lignocellulose biomass and extensively used in bioprocess and fermentation technology and many species are also a generally regarded safe. Many of the Aspergillus species are established cell factories due to their inherent capacity in secreting large number of hydrolytic enzymes. With the advent of next generation genomic technologies and metabolic engineering technologies, the production potential of Aspergillus cell factory has improved over the years. Various genome editing tools has been developed for Aspergillus like engineered nucleases, zinc finger nucleases, TALEN and CRISPR-Cas9 system. Currently, the CRISPR/Cas9-based technique is extensively used to enhance the effectiveness of gene manipulation in model system Aspergillus nidulans and other strains like Aspergillus oryzae, Aspergillus niger and Aspergillus fumigatus. This review describes the recent developments of genome editing technologies in Aspergillus the synthesis of heterologous proteins and secondary metabolites in the Aspergillus species.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 μg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Influence of the expression vector and its elements on recombinant human prolactin synthesis in Escherichia coli; co-directional orientation of replication and transcription is highly critical

The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of recombinant human prolactin (hPRL). In previous work, based on experiments that were basically carried out in parallel with the present ones, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower here (2.5–4-fold), in the RB791 and RRI strains. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~ 5–6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic background that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.

Thermal treatment improves a process of crude glycerol valorization for the production of a heterologous enzyme by Yarrowia lipolytica

Valorization of crude glycerol requires a potent bifunctional biocatalyst, such as Yarrowia lipolytica, capable of high-density growth on this substrate, and having i.a. high propensity for heterologous protein synthesis. Increasing evidence suggests that controlled administration of stress, i.a. thermal treatment, has a positive impact on bioprocess performance. In this study, we systematically adjusted thermal treatment conditions (20 to 42 °C) in order to maximize heterologous protein production by Y. lipolytica growing in crude glycerol-based medium. Our results showed nearly 30% enhancement in the enzyme production triggered by temporary exposure to decreased temperature. Here developed mathematical model indicated optimal treatment conditions (20 °C, 153′) that were later applied to a process with biodiesel-derived glycerol and technical substrates. Techno-economic analysis of a pilot-scale-waste-free process was conducted. Quantitative description of the associated costs and economic gain due to exploitation of industrial substrates, as well as indication of current bottlenecks of the process, are also provided.

Post‐Translational Modifications Regulate Lipid Remodeling Enzyme During Freezing Stress

As winter approaches, plants acclimate to the cold and begin to induce physiological and metabolic changes that are necessary for survival. While we know much about the signaling pathways that induce metabolic changes at the onset of cold and early freezing, less is known about signaling during severe freezing. One such signaling pathway is the activation of the cryoprotective enzyme Sensitive to Freezing 2 (SFR2). Even though SFR2 is constitutively expressed, it only becomes active as the plant experiences subzero temperatures. The function of SFR2 is to recognize these freezing temperatures and induce membrane remodeling to produce oligogalactolipids, lipids containing two or more galactose molecules as a head group. Because of its ability to sense freezing, many cold-tolerant species including the model plant Arabidopsis thaliana, require SFR2 activity to survive more severe freezing temperatures. However, the signaling pathway that enables SFR2 to determine when temperatures reach freezing and to become active remains unknown. Here we show that SFR2 activity is regulated by post-translational modifications, specifically phosphorylations, and that these are the mechanism by which SFR2 can sense a freezing stress. To determine this, we took two complementary approaches. The first used Arabidopsis thaliana exposed to control, cool or freezing conditions. Following temperature treatment, SFR2 was immunoprecipitated and mass spectrometry measurements were taken. From this, we were able to determine a unique phosphorylation pattern on SFR2 at each temperature analyzed. To assess the importance of phosphorylations on the residues of interest we used site-directed mutagenesis on SFR2 to systematically change the amino acids we found to be phosphorylated to alanine. The effect of these mutations was measured using our second approach, a heterologous expression system in which SFR2 is always active. Functional assays of the SFR2 mutants in this system showed that the residues identified via mass spectrometry were only a portion of those needed for activity. We confirmed this by designing two unique non-phosphorylatable SFR2s. This was accomplished with in silico modeling of likely phosphorylatable residues, followed by mutagenesis to alanine. Heterologous protein synthesis and function analysis of these non-phosphorylatable SFR2s suggest these mutants are unable to synthesize oligogalactolipids and are currently being assessed in planta. Using both the Arabidopsis and heterologous expression data we will identify the specific residues required for SFR2 activation and the kinases responsible by determining recognition motifs surrounding the residues. By understanding how metabolic signals change during freezing stress, we can elucidate the dynamics surrounding low-temperature enzyme activation and direct research for increased cold tolerance in crops.

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins.

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well‐defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.

Novel Expression Vectors Based on the pIGDM1 Plasmid

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIGDM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIGDMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals.

Efficient transformation of the entomopathogenic fungus Lecanicillium muscarium by electroporation of germinated conidia

Abstract All experiments on genetic modifications of Lecanicillium species performed so far used laborious PEG-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. In this study we demonstrated that simple and effective method of electroporation of germinated conidia can be used for transformation of this fungus. Electroporation of L. muscarium (strain VL 72) germinated conidia by the pBARGPE1 vector harboring an eGFP gene, showed a transformation efficiency 65.7 ± 1.4 phosphinothricin-resistant colonies per 2.5 μg of linearized plasmid DNA and expression of fluorescent protein without affecting fungal growth and virulence. By analysis of GFP-expressing isolates by fluorescent microscopy and immunoblotting, we found that the Aspergillus nidulans PgpdA promoter and trpC terminator in the pBARGPE1 vector provide an effective synthesis of heterologous proteins in L. muscarium cells.

Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

BackgroundTranslational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes.ResultsHere, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation.ConclusionGlobal decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins.

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.

Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein

BackgroundThe Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and ^{13}hbox {C}-based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein.ResultsRNA-seq and ^{13}hbox {C}-based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion.ConclusionsTranscriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.

Expression of Ac-PK2 protein from AcMNPV improved the progeny virus production via regulation of energy metabolism and protein synthesis.

Baculovirus encoded PK2 protein can increase viral fitness through inhibition of the eIF2α family kinases activity. Previous studies indicated that the virus might take over the control of cellular machinery post-infection, which would impose a high metabolic burden to infected insect cells. Here we showed that eIF2α phosphorylation decreased, with concomitant up-regulation of total and heterologous protein synthesis in AcMNPV-PK2-EGFP infected Sf9 cells and the larvae of Spodoptera exigua. Simultaneously, the lactic acid accumulation decreased and the uptake of glucose increased in AcMNPV-PK2-EGFP infected Sf9 cells. We proposed a model that Ac-PK2 protein overexpression would help protein synthesis by inhibiting eIF2α phosphorylation, which provided a more favorable scenario to support the efficient replication of the virus by re-directing the cellular metabolism toward ATP production. Finally, we confirmed that AcMNPV-PK2-EGFP could improve the production of progeny virus in infected Sf9 cells and enhance insecticidal activity against Spodoptera exigua larvae.

Quantitative physiology and elemental composition of Kluyveromyces lactis CBS 2359 during growth on glucose at different specific growth rates.

The yeast Kluyveromyces lactis has received attention both from academia and industry due to some important features, such as its capacity to grow in lactose-based media, its safe status, its suitability for large-scale cultivation and for heterologous protein synthesis. It has also been considered as a model organism for genomics and metabolic regulation. Despite this, very few studies were carried out hitherto under strictly controlled conditions, such as those found in a chemostat. Here we report a set of quantitative physiological data generated during chemostat cultivations with the K. lactis CBS 2359 strain, obtained under glucose-limiting and fully aerobic conditions. This dataset serves [corrected] as a basis for the comparison of K. lactis with the model yeast Saccharomyces cerevisiae in terms of their elemental compositions, as well as for future metabolic flux analysis and metabolic modelling studies with K. lactis.

The importance of arginine codons AGA and AGG for the expression in E. coli of triosephosphate isomerase from seven different species

Rare arginine codons AGA and AGG affect the heterologous expression of proteins in Eschericha coli. The tRNAs necessary for protein synthesis are scarce in E. coli strain BL21(DE3) pLysS and plentiful in strain BL21(DE3) CodonPlus −RIL. We evaluated in both bacterial strains the effect of these rare codons on the expression of triosephosphate isomerases from 7 different species, whose sequences had different dispositions of rare arginine codons. The ratio of expressed protein (CP/Bl21) correlated with the number of rare codons. Our study shows that the number, position and particularities of the combination of rare Arg codons in the natural non-optimized sequences of the triosephosphate isomerases influence the synthesis of heterologous proteins in E. coli and could have implications in the selection of better sequences for engineering enzymes for novel or manipulated metabolic pathways or for the expression levels of non enzymatic proteins..

Kluyveromyces marxianus as a host for heterologous protein synthesis.

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10°C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.