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Methanol is an ideal feedstock for chemical and biological manufacturing. Constructing an efficient cell factory is essential for producing complex compounds through methanol biotransformation, in which coordinating methanol use and product synthesis is often necessary. In methylotrophic yeast, methanol utilization mainly occurs in peroxisomes, which creates challenges in driving the metabolic flux toward product biosynthesis. Here, we observed that constructing the cytosolic biosynthesis pathway resulted in compromised fatty alcohol production in the methylotrophic yeast Ogataea polymorpha. Alternatively, peroxisomal coupling of fatty alcohol biosynthesis and methanol utilization significantly improved fatty alcohol production by 3.9-fold. Enhancing the supply of precursor fatty acyl-CoA and cofactor NADPH in the peroxisomes by global metabolic rewiring further improved fatty alcohol production by 2.5-fold and produced 3.6 g/L fatty alcohols from methanol under fed-batch fermentation. We demonstrated that peroxisome compartmentalization is helpful for coupling methanol utilization and product synthesis, and with this approach, constructing efficient microbial cell factories for methanol biotransformation is feasible.
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Alcoholes Grasos , Metanol , Alcoholes Grasos/metabolismo , Metanol/metabolismo , Peroxisomas/metabolismo , Fermentación , Ingeniería Metabólica/métodosRESUMEN
Methanol, a rich one-carbon feedstock, can be massively produced from CO2 by the liquid sunshine route, which is helpful to realize carbon neutrality. ß-Farnesene is widely used in the production of polymers, surfactants, lubricants, and also serves as a suitable substitute for jet fuel. Constructing an efficient cell factory is a feasible approach for ß-farnesene production through methanol biotransformation. Here, we extensively engineered the methylotrophic yeast Ogataea polymorpha for the efficient bio-production of ß-farnesene using methanol as the sole carbon source. Our study demonstrated that sufficient supply of precursor acetyl-CoA and cofactor NADPH in an excellent yeast chassis had a 1.3-fold higher ß-farnesene production than that of wild-type background strain. Further optimization of the mevalonate pathway and enhancement of acetyl-CoA supply led to a 7-fold increase in ß-farnesene accumulation, achieving the highest reported sesquiterpenoids production (14.7 g/L with a yield of 46 mg/g methanol) from one-carbon feedstock under fed-batch fermentation in bioreactor. This study demonstrates the great potential of engineering O. polymorpha for high-level terpenoid production from methanol.
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Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.
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Productos Biológicos , Saccharomyces cerevisiae , Productos Biológicos/metabolismo , Ácidos Cafeicos/metabolismo , Hidroxibenzoatos , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : ⢠22 promoters related to methanol metabolism were characterized in O. polymorpha. ⢠Promoter truncation resulted shorter and compact promoters. ⢠Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.
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Ingeniería Metabólica , Metanol , Pichia/genética , Regiones Promotoras Genéticas , SaccharomycetalesRESUMEN
BACKGROUND: Currently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified. RESULTS: A complex of SLA-I constituted by an SLA-2*HB01 molecule with swine ß2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64-72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å. CONCLUSION: This research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.
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Epítopos , Virus de la Fiebre Aftosa/genética , Antígenos de Histocompatibilidad Clase I , Modelos Moleculares , Difracción de Rayos X , Animales , Cristalización , Epítopos/química , Epítopos/genética , Epítopos/aislamiento & purificación , Epítopos/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Serogrupo , Porcinos , Linfocitos T Citotóxicos/metabolismoRESUMEN
Methylotrophic yeast Ogataea polymorpha has become a promising cell factory due to its efficient utilization of methanol to produce high value-added chemicals. However, the low homologous recombination (HR) efficiency in O. polymorpha greatly hinders extensive metabolic engineering for industrial applications. Overexpression of HR-related genes successfully improved HR efficiency, which however brought cellular stress and reduced chemical production due to constitutive expression of the HR-related gene. Here, we engineered an HR repair pathway using the dynamically regulated gene ScRAD51 under the control of the l-rhamnose-induced promoter PLRA3 based on the previously constructed CRISPR-Cas9 system in O. polymorpha. Under the optimal inducible conditions, the appropriate expression level of ScRAD51 achieved up to 60% of HR rates without any detectable influence on cell growth in methanol, which was 10-fold higher than that of the wild-type strain. While adopting as the chassis strain for bioproductions, the dynamically regulated recombination system had 50% higher titers of fatty alcohols than that static regulation system. Therefore, this study provided a feasible platform in O. polymorpha for convenient genetic manipulation without perturbing cellular fitness.
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Construction of efficient microbial cell factories always requires assembling biosynthetic pathways and rewiring cellular metabolism with overexpression of multiple genes. Genomic integration is considered to be helpful for stable gene expression in compared with the episomal plasmids. However, the limited availability of suitable loci hinders the extensive metabolic engineering. We here characterized 30 neutral sites in Saccharomyces cerevisiae genome that did not affect cellular fitness by using expression cassettes of green fluorescent protein (eGFP) and fatty acyl-CoA reductase (MaFAR1) with the aid of efficient CRISPR-Cas9 technique. We found that integration of gene expression cassettes to different genome loci resulted a varied GFP signal and fatty alcohol production, which showed that genomic loci could be used for tuning gene expression. The characterized set of neutral sites should be helpful for extensively metabolic engineering of S. cerevisiae for chemical production and other purposes.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sistemas CRISPR-Cas , Expresión Génica , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P PDH , P PYK , P FBA , P PGM , P GLK , P TRI , P GPI , P ADH1 , P TEF1 and P GCW14 ) were obtained with the activity range of 13%-130% of the common promoter P GAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P PDH and P GCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P ICL1 , rhamnose-induced P LRA3 and P LRA4 , and a bidirectional promoter (P Mal -P Per ) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P LRA3 by 4.7-10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.
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α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals.
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Energy shortage and environmental concern urgently require establishing the feasible bio-refinery process from various feedstocks. The methylotrophic yeast Ogataea polymorpha is thermo-tolerant and can utilize various carbon sources, such as glucose, xylose and methanol, which makes it a promising host for bio-manufacturing. Here, we explored the capacity of O. polymorpha for overproduction of free fatty acids (FFAs) from multiple substrates. The engineered yeast produced 674 mg/L FFA from 20 g/L glucose in shake flask and could sequentially utilize the mixture of glucose and xylose. However, the FFA producing strain failed to survive in sole methanol and supplementing co-substrate xylose promoted methanol metabolism. A synergistic utilization of xylose and methanol was observed in the FFA producing strain. Finally, a mixture of glucose, xylose and methanol was evaluated for FFA production (1.2 g/L). This study showed that O. polymorpha is an ideal host for chemical production from various carbon sources.
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Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy, making it an ideal host for bio-manufacturing. However, the lack of gene integration sites limits its systems metabolic engineering, in particular construction of genome-integrated pathway. We here screened the genomic neutral sites for gene integration without affecting cellular fitness, by genomic integration of an enhanced green fluorescent protein (eGFP) gene via CRISPR-Cas9 technique. After profiling the growth and fluorescent intensity in various media, 17 genome positions were finally identified as potential neutral sites. Finally, integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3, enabled the production of 4.5 mg/L fatty alcohols, indicating that these neutral sites can be used for streamline metabolic engineering in O. polymorpha. We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.
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Methanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%-30% to 60%-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the production of fatty alcohols in O. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.
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Currently available vaccines from inactivated foot-and-mouth disease virus (FMDV) only protect animals by inducing neutralizing antibodies. A vaccine that contains cytotoxic T lymphocytes (CTL) epitopes to induce strong CTL responses might protect animals more effectively. Herein, we used swine leukocyte antigen class I (SLAI) proteins derived from six different strains of domestic pigs to screen and identify shared FMDV CTL epitopes. Four potential FMDV CTL epitopes (Q01, Q02, AS3, and QA4) were confirmed by mass spectrometry. We also determined the antigenicity of these epitopes to elicit cell-mediated immunoresponse by the ELISPOT and CTL assays. Among the four peptides, Q01 and QA4 were found to bind all six SLA-I proteins with strong affinity and elicit significant activity of CTL (Pâ¯<â¯0.01). We conclude that Q01 and QA4 peptides are novel shared epitopes that can be recognized by all six SLA-I molecules on representative CTLs.
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Epítopos de Linfocito T/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Escherichia coli , Masculino , Unión Proteica , Especificidad de la Especie , PorcinosRESUMEN
Up to now, no crystal structure of swine leukocyte antigen 2 (SLA-2) molecules was reported. In order to elucidate the structure of SLA-2 and to study the cytotoxic T lymphocyte (CTL) epitopes derived from foot-and-mouth disease virus (FMDV), a complex of swine major histocompatibility complex (MHC) class I molecule (SLA-2 haplotype, Hebao allele) with swine ß2-microglobulin and the CTL epitope FMDV-Hu64 (ALLRTATYY) derived from O serotype of FMDV VP1 protein (residues 64-72) was refolded and crystallized. The crystal, which belonged to space group P212121, diffracted to 2.5â¯Å resolution and had unit cell parameters aâ¯=â¯48.37, bâ¯=â¯97.75, câ¯=â¯166.163â¯Å. These results will help to determine the first structure of a SLA-2 molecule in the context of an FMDV CTL epitope.