Improving functional annotation for industrial microbes: a case study with Pichia pastoris.
Ontology highlight
ABSTRACT:
SUBMITTER: Dikicioglu D
PROVIDER: S-EPMC4111905 | biostudies-literature | 2014 Aug
REPOSITORIES: biostudies-literature
Publications
Improving functional annotation for industrial microbes: a case study with Pichia pastoris.
Dikicioglu Duygu D Wood Valerie V Rutherford Kim M KM McDowall Mark D MD Oliver Stephen G SG
Trends in biotechnology 20140611 8
The research communities studying microbial model organisms, such as Escherichia coli or Saccharomyces cerevisiae, are well served by model organism databases that have extensive functional annotation. However, this is not true of many industrial microbes that are used widely in biotechnology. In this Opinion piece, we use Pichia (Komagataella) pastoris to illustrate the limitations of the available annotation. We consider the resources that can be implemented in the short term both to improve G ...[more]
Similar Datasets
Project description:The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68-90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6-380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.
| S-EPMC8287956 | biostudies-literature
Project description:BackgroundMethanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.ResultsIn the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine).ConclusionsThis study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.
| S-EPMC10540378 | biostudies-literature
Project description:BackgroundPhytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.MethodsTo improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.ResultsThe phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.DiscussionThe production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.ConclusionsUsing modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.
| S-EPMC4584009 | biostudies-literature
Project description:BackgroundThe effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed.ResultsIn-depth growth profiling of evolved populations revealed several instances of genotype-dependent growth trade-off/cross-benefit correlations in non-evolutionary growth conditions. On the genome level a high degree of mutational convergence was observed among independent populations. Environment-dependent mutational hotspots were related to osmotic stress-, Rim - and cAMP signaling pathways. In agreement with the observed growth phenotypes, our data also suggest diverging compensatory mutations in glycosylation-deficient populations. High osmolarity glycerol (HOG) pathway loss-of-functions mutations, including genes such as SSK2 and SSK4, represented a major adaptive strategy during environmental adaptation. However, genotype-specific HOG-related mutations were predominantly observed in opposing environmental conditions. Surprisingly, such mutations emerged during salt stress adaptation in OCH1 knockout populations and led to growth trade-offs in non-adaptive conditions that were distinct from wildtype HOG-mutants. Further environment-dependent mutations were identified for a hitherto uncharacterized species-specific Gal4-like transcriptional regulator involved in environmental sensing.ConclusionWe show that metabolic constraints such as glycosylation-deficiency can contribute to evolution on the molecular level, even in non-diverging growth environments. Our dataset suggests universal adaptive mechanisms involving cellular stress response and cAMP/PKA signaling but also the existence of highly species-specific strategies involving unique transcriptional regulators, improving our biological understanding of distinct Ascomycetes species.
| S-EPMC5553748 | biostudies-literature
Project description:Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory.A blood-active laccase (ChU-B mutant) fused to the native/evolved ?-factor prepro-leader was cloned under the control of two different promoters (P(AOX1) and P(GAP)) and expressed in Pichia pastoris. The most active construct, which contained the P(AOX1) and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the ?-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers.The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.
| S-EPMC3655043 | biostudies-literature
Project description:Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1-S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.
| S-EPMC3935203 | biostudies-literature
Project description:Comparison of transcription profile of Pichia pastoris cells grown on Glucose medium with Pichia pastoris cells grown on Methanol/Glycerol medium, the fermentations were done in a chemostat.
2015-08-31 | GSE67690 | GEO
Project description:α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.
| S-EPMC4993465 | biostudies-literature
Project description:Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the ?12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.
| S-EPMC4617397 | biostudies-literature
Project description: Not available
| S-EPMC10991172 | biostudies-literature