A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

All authors are with the Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA -- Hal S. Alper is with the Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA -- Amanda M. Lanza Current Address: Bristol-Myers Squibb, Biologics Development, 35 South Street, Hopkinton, MA 01748, USABackground: Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results: Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions: Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.Chemical EngineeringInstitute for Cellular and Molecular [email protected]

Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems.Materials and Methods: To express LmSTI1 in the methylotrophic yeast         Pichia pastoris (P. pastoris), the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively.Results: The expression level was 0.2% of total soluble proteins.Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Synthetic gene design - The rationale for codon optimization and implications for molecular pharming in plants.

Degeneracy in the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias in genes is the term given to the preferred use of particular synonymous codons. Synonymous codon substitutions had been regarded as "silent" as the primary structure of the protein was not affected; however, it is now accepted that synonymous substitutions can have a significant effect on heterologous protein expression. Codon optimization, the process of altering codons within the gene sequence to improve recombinant protein expression, has become widely practised. Multiple inter-linked factors affecting protein expression need to be taken into consideration when optimizing a gene sequence. Over the years, various computer programmes have been developed to aid in the gene sequence optimization process. However, as the rulebook for altering codon usage to affect protein expression is still not completely understood, it is difficult to predict which strategy, if any, will design the 'optimal' gene sequence. In this review, codon usage bias and factors affecting codon selection will be discussed and the evidence for codon optimization impact will be reviewed for recombinant protein expression using plants as a case study. These developments will be relevant to all recombinant expression systems, however, molecular pharming in plants is an area which has consistently encountered difficulties with low levels of recombinant protein expression, and should benefit from an evidence based rational approach to synthetic gene design

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Characterization of DNA polymerase gamma in the basidiomycetous yeast, Cryptococcus neoformans

Cryptococcus neoformans is a common basidiomycetous yeast and an obligate aerobe that inhabits most environments across the world. C. neoformans is also an opportunistic pathogencapable of causing a fungal infection of the central nervous system known as cryptococcal meningoencephalitis. This disease is often observed in patients suffering from AIDS or other immune-compromising afflictions. The antifungal medications currently used to treat C. neoformans infections are expensive and often incapable of eradicating infection. Understanding vital systems in this organism are critical to the production of cheaper and more effective medications. Mitochondria are a critically important organelle in aerobic organisms, generating more than 90% of cellular ATP. DNA polymerase gamma (PolG) is a nuclear-encoded DNA polymerase that has been shown in many organisms like humans, Drosophila, and Saccharomyces, to play a vital role in maintaining and replicating mitochondrial DNA. Homology searches have revealed that C. neoformans contains a single putative DNA polymerase gamma gene (CnMIP1). The aims of this project were to determine the essentiality of this gene, and to characterize the biochemical function of the encoded protein (CnPolG). In order to determine if CnMIP1 was essential to the survival of this yeast, the gene was knocked down in vivo using RNA interference. This showed that cryptococci are incapable of survival without CnPolG, thus providing evidence of the importance of CnMIP1 in C. neoformans viability. Although DNA sequence homology showed the presence of a polymerase and an exonuclease domain in CnMIP1, the function of the encoded protein had to be experimentally determined. For this, the CnMIP1 gene was cloned and recombinant protein was expressed in both bacterial (pET28 plasmid) and yeast (pRS424 plasmid) expression systems. The bacterially expressed protein was heavily degraded, but addition of ethanol to the media has shown promising results. The yeast-produced protein, using a codon-optimized gene construct, showed significantly reduced levels of degradation, but this has been an unreliable expression system for large-scale production of this protein thus far. The polymerization ability of recombinant CnPolG purified from bacteria has been assayed using M13 ssDNA as template. Additionally, it has been shown that this protein can utilize either tailed or untailed primers to initiate polymerization and performs more robustly in a low salt environment. Finally, these assays have also demonstrated that polymerase activity is dependent on Mg2+ or Mn2+ ions and that functionality increases when both cations are present. Comparing homologues from Saccharomyces, humans, and Cryptococcus shows the presence of two domains in CnPolG that are not present in the other two homologues. Moving forward, these domains could provide functional insights into the regulation of mitochondrial polymerases and may serve as potential pharmaceutical targets if they play an essential role in CnMIP1 functionality

Engineering yeast for improved recombinant protein production

Recombinant proteins are broadly used from basic research to therapeutic development and include industrial enzymes and pharmaceutical proteins. The increasing demand for improved production and enhanced quality of recombinant proteins requires robust biotech-based strategies to overcome the limitations of protein extraction from natural sources. A variety of cell factories are therefore established for the large-scale production of recombinant proteins of interest. In comparison to other expression systems, the budding yeast Saccharomyces cerevisiae is an attractive production platform due to its high tolerance to harsh fermentation conditions, and importantly its capability to perform eukaryotic post-translational modifications and to secrete the biologically active product to the extracellular medium. Thus, many strategies have been applied to engineer this organism for increasing its recombinant protein secretory capacity and productivity.The major aim of this thesis work was to study and develop efficient yeast platforms for the production of different heterologous proteins for medical or industrial use through diverse engineering strategies. The first part of this work explored in depth a line of previously evolved yeast strains with improved protein secretory capacity. The universal applicability of the evolved strains was evaluated to produce different antibody fragments, but it was concluded that this secretion platform was not suitable for all types of pharmaceutical proteins tested. Furthermore, by re-introducing all 42 protein-sequence-altering mutations identified in the evolved strains into the parental strain using the CRISPR/Cas9 technology, 14 targets were shown to be beneficial for protein production and 11 out of these 14 beneficial targets were newly identified to be related to recombinant protein production. The second part of this work focused on investigating novel targets related to the cellular stress response and the protein secretory process to rationally optimize S. cerevisiae. Furthermore, screening for suppressors of amyloid-β cytotoxicity in a yeast Alzheimer’s disease model revealed a number of gene targets that reduced oxidative stress and improved production of recombinant proteins. Additionally, a proteome-constrained genome-scale protein secretory model of S. cerevisiae (pcSecYeast) was constructed to simulate the secretion of various recombinant proteins and predict system-level engineering targets for increasing protein production. In summary, the work presented in this thesis provides different efficient strategies to develop yeast platforms for the high-level production of valuable industrial or pharmaceutical proteins, and also provides general guidelines for designing other cell platforms for efficient protein production. Integrated application of various engineering approaches will make meaningful advancements in the field of recombinant protein production in the future

Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds

Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds.Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 202