RESUMO
Biochemistry of carbon assimilation in aerobic methylotrophs growing on reduced C1 compounds has been intensively studied due to the vital role of these microorganisms in nature. The biochemical pathways of carbon assimilation in methylotrophs growing on multi-carbon substrates are insufficiently explored. Here we elucidated the metabolic route of mannitol assimilation in the alphaproteobacterial facultative methylotroph Methylobrevis pamukkalensis PK2. Two key enzymes of mannitol metabolism, mannitol-2-dehydrogenase (MTD) and fructokinase (FruK), were obtained as His-tagged proteins by cloning and expression of mtd and fruK genes in Escherichia coli and characterized. Genomic analysis revealed that further transformation of fructose-6-phosphate proceeds via the Entner-Doudoroff pathway. During growth on mannitol + methanol mixture, the strain PK2 consumed both substrates simultaneously demonstrating independence of C1 and C6 metabolic pathways. Genome screening showed that genes for mannitol utilization enzymes are present in other alphaproteobacterial methylotrophs predominantly capable of living in association with plants. The capability to utilize a variety of carbohydrates (sorbitol, glucose, fructose, arabinose and xylose) suggests a broad adaptability of the strain PK2 to live in environments where availability of carbon substrate dynamically changes.
Assuntos
Frutoquinases , Manitol , Manitol/metabolismo , Frutoquinases/metabolismo , Frutoquinases/genética , Manitol Desidrogenases/metabolismo , Manitol Desidrogenases/genética , Frutosefosfatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Redes e Vias Metabólicas/genética , Metanol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimentoRESUMO
1, 3-propanediol (1, 3-PDO) is an important diol with wide applications in the pharmaceutical, food, and cosmetics industries. In addition, 1, 3-PDO serves as a crucial monomer in the synthesis of polytrimethylene terephthalate, an important synthetic fiber material. Microbial conversion of renewable resources such as glucose into 1, 3-PDO has been industrialized due to its environmentally friendly, energy-efficient, safe, and sustainable characteristics. It serves as a successful case in the design and application of microbial cell factories for biochemicals. However, concerns such as food scarcity and climate change are driving the exploration of non-food, low-cost, and sustainable alternatives as biomanufacturing feedstocks. The biosynthesis of 1, 3-PDO from the C3 feedstock glycerol by microorganisms has been well studied. In recent years, increasing attention has been paid to the synthesis of 1, 3-PDO from C1 feedstocks such as methanol, which has higher energy density than glucose and glycerol. Several new artificial biosynthetic pathways have been proposed and validated, laying a foundation for the sustainable bioproduction of 1, 3-PDO. This article reviews the feedstock transition from C6 to C3 and C1 carbon sources for the microbial synthesis of 1, 3-PDO and discusses the strategies for reprogramming metabolic pathway to enhance 1, 3-PDO biosynthesis from different feedstocks. Finally, the development prospects of 1, 3-PDO bioproduction from C1 feedstocks are forecasted.
Assuntos
Carbono , Propilenoglicóis , Carbono/metabolismo , Propilenoglicóis/metabolismo , Glicerol/metabolismo , Microbiologia Industrial , Glucose/metabolismo , Engenharia Metabólica , Metanol/metabolismo , Vias Biossintéticas , Fermentação , Bactérias/metabolismoRESUMO
Methanol has been considered one of the most important alternative carbon sources for the next-generation biomanufacturing due to its low price, mature production processes, and potential sustainability. Constructing microbial cell factories for methanol to chemical biotransformation has become a research hotspot in the green biomanufacturing industry. Focusing on the microorganisms that can naturally use methanol, we compare them with non-natural cell factories for chemical production from methanol. We discuss the key issues and challenges associated with natural cell factories for chemical production from methanol, summarize recent research progress surrounding these issues, and propose possible solutions to these challenges. This review helps to generate feasible guidelines and research strategies for the modification of natural cell factories for efficient methanol to chemical production in the future.
Assuntos
Microbiologia Industrial , Metanol , Metanol/metabolismo , Microbiologia Industrial/tendências , Biotransformação , Bactérias/metabolismo , Engenharia MetabólicaRESUMO
Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO2 and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of ß-farnesene from dual carbon source by replacing the α-bisabolene synthase with a ß-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.
Assuntos
Glucose , Engenharia Metabólica , Metanol , Sesquiterpenos , Metanol/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Sesquiterpenos/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Ácido Mevalônico/metabolismoRESUMO
The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.
Assuntos
Metanol , Methylobacterium , Methylobacterium/metabolismo , Methylobacterium/genética , Methylobacterium/enzimologia , Methylobacterium/crescimento & desenvolvimento , Metanol/metabolismo , Simbiose , Mutação , Aldeído Liases/metabolismo , Aldeído Liases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Folhas de Planta/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Methylobacterium extorquens/crescimento & desenvolvimento , Methylobacterium extorquens/enzimologia , Desenvolvimento Vegetal , Microbiota/genética , BiomassaRESUMO
BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.
Assuntos
Engenharia Metabólica , Metanol , Saccharomycetales , Metanol/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/genética , Engenharia Metabólica/métodos , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Formaldeído/metabolismo , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , NAD/metabolismoRESUMO
BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.
Assuntos
Reatores Biológicos , Oxigenases de Função Mista , Regiões Promotoras Genéticas , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Metanol/metabolismoRESUMO
Cupriavidus metallidurans strain PD11 isolated from laboratory waste drainage can use C1 compounds, such as dichloromethane (DCM) and methanol, as a sole carbon and energy source. However, strain CH34 (a type-strain) cannot grow in the medium supplemented with DCM. In the present study, we aimed to unravel the genetic elements underlying the utilization of C1 compounds by strain PD11. The genome subtraction approach indicated that only strain PD11 had several genes highly homologous to those of Herminiimonas arsenicoxydans strain ULPAs1. Moreover, a series of polymerase chain reaction (PCR) to detect the orthologs of H. arsenicoxydans genes and the comparative study of the genomes of three strains revealed that the 87.9 kb DNA fragment corresponding to HEAR1959 to HEAR2054 might be horizontally transferred to strain PD11. The 87.9 kb DNA fragment identified was found to contain three genes whose products were putatively involved in the metabolism of formaldehyde, a common intermediate of DCM and methanol. In addition, reverse transcription PCR analysis showed that all three genes were significantly expressed when strain PD11 was cultivated in the presence of DCM or methanol. These findings suggest that strain PD11 can effectively utilize the C1 compounds because of transfer of the mobile genetic elements from other bacterial species, for instance, from H. arsenicoxydans.
Assuntos
Cupriavidus , Sequências Repetitivas Dispersas , Metanol , Cloreto de Metileno , Metanol/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Cupriavidus/efeitos dos fármacos , Cloreto de Metileno/metabolismo , Sequências Repetitivas Dispersas/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Genoma Bacteriano/genética , Transferência Genética HorizontalRESUMO
A bioelectrochemical upflow anaerobic sludge blanket (BE-UASB) was constructed and compared with the traditional UASB to investigate the role of bioelectrocatalysis in modulating methanogenesis and sulfidogensis involved within anaerobic treatment of high-sulfate methanolic wastewater (COD/SO42- ratio ≤ 2). Methane production rate for BE-UASB was 1.4 times higher than that of the single UASB, while SO42- removal stabilized at 16.7%. Bioelectrocatalysis selectively enriched key functional anaerobes and stimulated the secretion of extracellular polymeric substances, especially humic acids favoring electron transfer, thereby accelerating the electroactive biofilms development of electrodes. Methanomethylovorans was the dominant genus (35%) to directly convert methanol to CH4. Methanobacterium as CO2 electroreduction methane-producing archaea appeared only on electrodes. Acetobacterium exhibited anode-dependence, which provided acetate for sulfate-reducing bacteria (norank Syntrophobacteraceae and Desulfomicrobium) through synergistic coexistence. This study confirmed that BE-UASB regulated the microbial ecology to achieve efficient removal and energy recovery of high-sulfate methanolic wastewater.
Assuntos
Metano , Metanol , Esgotos , Sulfatos , Águas Residuárias , Águas Residuárias/microbiologia , Metanol/metabolismo , Metano/metabolismo , Sulfatos/metabolismo , Esgotos/microbiologia , Anaerobiose , Reatores Biológicos/microbiologia , EletrodosRESUMO
Methanol is a promising feedstock for the bio-based economy as it can be derived from organic waste streams or produced electrochemically from CO2. Acetate production from CO2 in microbial electrosynthesis (MES) has been widely studied, while more valuable compounds such as butyrate are currently attracting attention. In this study, methanol was used as a co-substrate with CO2 to enhance butyrate production in MES. Feeding with CO2 and methanol resulted in the highest butyrate production rates and titres of 0.36 ± 0.01 g L-1 d-1 and 8.6 ± 0.2 g L-1, respectively, outperforming reactors with only CO2 feeding (0.20 ± 0.03 g L-1 d-1 and 5.2 ± 0.1 g L-1, respectively). Methanol acted as electron donor and as carbon source, both of which contributed ca. 50% of the carbon in the products. Eubacterium was the dominant genus with 52.6 ± 2.5% relative abundance. Thus, we demonstrate attractive route for the use of the C1 substrates, CO2 and methanol, to produce mainly butyrate. KEY POINTS: ⢠Butyrate was the main product from methanol and CO2 in MES ⢠Methanol acted as both carbon and electron source in MES ⢠Eubacterium dominating microbial culture was enriched in MES.
Assuntos
Butiratos , Dióxido de Carbono , Metanol , Metanol/metabolismo , Dióxido de Carbono/metabolismo , Butiratos/metabolismo , Reatores Biológicos/microbiologia , Carbono/metabolismo , Acetatos/metabolismoRESUMO
d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.
Assuntos
Escherichia coli , Fermentação , Metanol , RNA Antissenso , Xilose , Escherichia coli/genética , Escherichia coli/metabolismo , Xilose/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , Metanol/metabolismo , Engenharia Metabólica , Frutose/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Microbes that use the single-carbon substrates methanol and methane offer great promise to bioindustry along with substantial environmental benefits. Methanotrophs and other methylotrophs can be engineered and optimized to produce a wide range of products, from biopolymers to biofuels and beyond. While significant limitations remain, including delivery of single-carbon feedstock to bioreactors, efficient growth, and scale-up, these challenges are being addressed and notable improvements have been rapid. Development of expression chassis, use of genome-scale and regulatory models based on omics data, improvements in bioreactor design and operation, and development of green product recovery schemes are enabling the rapid development of single-carbon bioconversion in the industrial space.
Assuntos
Reatores Biológicos , Metano , Metano/metabolismo , Reatores Biológicos/microbiologia , Bioengenharia/métodos , Biocombustíveis/microbiologia , Bactérias/metabolismo , Bactérias/genética , Microbiologia Industrial/métodos , Metanol/metabolismoRESUMO
Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.
Assuntos
Metano , Óxido Nitroso , Óxido Nitroso/metabolismo , Metano/metabolismo , Concentração de Íons de Hidrogênio , Oxirredutases/metabolismo , Oxirredutases/genética , Oxigênio/metabolismo , Oxirredução , Anaerobiose , Metanol/metabolismo , Hidrogênio/metabolismo , Oxigenases/metabolismo , Oxigenases/genéticaRESUMO
Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.
Assuntos
Metano , Methylocystaceae , Oryza , Rizosfera , Microbiologia do Solo , Oryza/microbiologia , Methylocystaceae/genética , Methylocystaceae/metabolismo , Methylocystaceae/isolamento & purificação , Metano/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , China , Metanol/metabolismoRESUMO
Methanogenic archaea play a key role in the global carbon cycle because these microorganisms remineralize organic compounds in various anaerobic environments. The microorganism Methanosarcina barkeri is a metabolically versatile methanogen, which can utilize acetate, methanol, and H2/CO2 to synthesize methane. However, the regulatory mechanisms underlying methanogenesis for different substrates remain unknown. In this study, RNA-seq analysis was used to investigate M. barkeri growth and gene transcription under different substrate regimes. According to the results, M. barkeri showed the best growth under methanol, followed by H2/CO2 and acetate, and these findings corresponded well with the observed variations in genes transcription abundance for different substrates. In addition, we identified a novel regulator, MSBRM_RS03855 (designated as HdrR), which specifically activates the transcription of the heterodisulfide reductase hdrBCA operon in M. barkeri. HdrR was able to bind to the hdrBCA operon promoter to regulate transcription. Furthermore, the structural model analyses revealed a helix-turn-helix domain, which is likely involved in DNA binding. Taken together, HdrR serves as a model to reveal how certain regulatory factors control the expression of key enzymes in the methanogenic pathway.IMPORTANCEThe microorganism Methanosarcina barkeri has a pivotal role in the global carbon cycle and contributes to global temperature homeostasis. The consequences of biological methanogenesis are far-reaching, including impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. As such, reducing methane emissions is crucial to meeting set climate goals. The methanogenic activity of certain microorganisms can be drastically reduced by inhibiting the transcription of the hdrBCA operon, which encodes heterodisulfide reductases. Here, we provide novel insight into the mechanisms regulating hdrBCA operon transcription in the model methanogen M. barkeri. The results clarified that HdrR serves as a regulator of heterodisulfide reductase hdrBCA operon transcription during methanogenesis, which expands our understanding of the unique regulatory mechanisms that govern methanogenesis. The findings presented in this study can further our understanding of how genetic regulation can effectively reduce the methane emissions caused by methanogens.
Assuntos
Proteínas Arqueais , Methanosarcina barkeri , Óperon , Oxirredutases , Methanosarcina barkeri/genética , Methanosarcina barkeri/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea , Transcrição Gênica , Metano/metabolismo , Metanol/metabolismo , Dióxido de Carbono/metabolismo , Acetatos/metabolismo , Hidrogênio/metabolismoRESUMO
Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha.
Assuntos
Metanol , Regiões Promotoras Genéticas , Proteínas Recombinantes , Saccharomycetales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Metanol/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
The development of synthetic microorganisms that could use one-carbon compounds, such as carbon dioxide, methanol, or formate, has received considerable interest. In this study, we engineered Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotrophy and formatotrophy, enabling them to co-utilize methanol or formate with CO2 fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consisted of a methanol-formate oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using endogenous enzymes, followed by blocking the native methanol assimilation pathway, modularly engineering genes of MFORG pathway, and compartmentalizing the methanol oxidation module. These modifications successfully enabled the methylotrophic yeast P. pastoris to utilize both methanol and formate. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing the synthetic methylotrophy and formatotrophy in this organism. The resulting strain could also successfully utilize both methanol and formate with consumption rates of 20 mg/L/h and 36.5 mg/L/h, respectively. The ability of the engineered P. pastoris and S. cerevisiae to co-assimilate CO2 with methanol or formate through the MFORG pathway was also confirmed by 13C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid by co-assimilating methanol and CO2 was demonstrated in the engineered P. pastoris and S. cerevisiae. This work indicates the potential of the MFORG pathway in developing different hosts to use various one-carbon compounds for chemical production.
Assuntos
Dióxido de Carbono , Formiatos , Engenharia Metabólica , Metanol , Saccharomyces cerevisiae , Formiatos/metabolismo , Metanol/metabolismo , Dióxido de Carbono/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/genéticaRESUMO
Perfluorooctane sulfonate (PFOS) is a prominent perfluorinated compound commonly found in the environment, known to pose various risks to human health. However, the removal of PFOS presents significant challenges, primarily due to the limited discovery of bacteria capable of effectively degrading PFOS. Moreover, single degradation bacteria often encounter obstacles in individual cultivation and the breakdown of complex pollutants. In contrast, microbial consortia have shown promise in pollutant degradation. This study employed a continuous enrichment method, combined with multiple co-metabolic substrates, to investigate a microbial consortium with the potential for PFOS degradation. By employing this methodology, we effectively identified a microbial consortium that demonstrated the capacity to reduce PFOS when exposed to an optimal concentration of methanol. The consortium predominantly comprised of Hyphomicrobium species (46.7%) along with unclassified microorganisms (53.0%). Over a duration of 20 days, the PFOS concentration exhibited a notable decrease of 56.7% in comparison to the initial level, while considering the exclusion of adsorption effects. Furthermore, by comparing the predicted metabolic pathways of the microbial consortium with the genome of a known chloromethane-degrading bacterium, Hyphomicrobium sp. MC1, using the KEGG database, we observed distinct variations in the metabolic pathways, suggesting the potential role of the unclassified microorganisms. These findings underscore the potential effectiveness of a "top-down" functional microbial screening approach in the degradation of stubborn pollutants.
Assuntos
Ácidos Alcanossulfônicos , Biodegradação Ambiental , Fluorocarbonos , Consórcios Microbianos , Fluorocarbonos/metabolismo , Ácidos Alcanossulfônicos/metabolismo , Bactérias/metabolismo , Bactérias/genética , Bactérias/classificação , Metanol/metabolismoRESUMO
Methanol, the second most abundant volatile organic compound, primarily released from plants, is a major culprit disturbing atmospheric chemistry. Interestingly, ubiquitously found methanol-utilizing bacteria, play a vital role in mitigating atmospheric methanol effects. Despite being extensively characterized, the effect of nitrogen sources on the richness of methanol-utilizers in the bulk soil and rhizosphere is largely unknown. Therefore, the current study was planned to isolate, characterize and explore the richness of cultivable methylotrophs from the bulk soil and rhizosphere of a paddy field using media with varying nitrogen sources. Our data revealed that more genera of methylotrophs, including Methylobacterium, Ancylobacter, Achromobacter, Xanthobacter, Moraxella, and Klebsiella were enriched with the nitrate-based medium compared to only two genera, Hyphomicrobium and Methylobacterium, enriched with the ammonium-based medium. The richness of methylotrophic bacteria also differed substantially in the bulk soil as compared to the rhizosphere. Growth characterization revealed that majority of the newly isolated methanol-utilizing strains in this study exhibited better growth at 37 °C instead of 30 or 45 °C. Moreover, Hyphomicrobium sp. FSA2 was the only strain capable of utilizing methanol even at elevated temperature 45 °C, showing its adaptability to a wide range of temperatures. Differential carbon substrate utilization profiling revealed the facultative nature of all isolated methanol-utilizer strains with Xanthobacter sp. TS3, being an important methanol-utilizer capable of degrading toxic compounds such as acetone and ethylene glycol. Overall, our study suggests the role of nutrients and plant-microbial interaction in shaping the composition of methanol-utilizers in terrestrial environment.
Assuntos
Bactérias , Metanol , Nitrogênio , Oryza , Rizosfera , Microbiologia do Solo , Nitrogênio/metabolismo , Metanol/metabolismo , Oryza/microbiologia , Bactérias/classificação , Bactérias/metabolismo , Bactérias/isolamento & purificação , Solo/química , RNA Ribossômico 16S/genética , Filogenia , Minerais/metabolismo , Temperatura , Carbono/metabolismoRESUMO
Soluble methane monooxygenase (sMMO) oxidizes a wide range of carbon feedstocks (C1 to C8) directly using intracellular NADH and is a useful means in developing green routes for industrial manufacturing of chemicals. However, the high-throughput biosynthesis of active recombinant sMMO and the ensuing catalytic oxidation have so far been unsuccessful due to the structural and functional complexity of sMMO, comprised of three functionally complementary components, which remains a major challenge for its industrial applications. Here we develop a catalytically active miniature of sMMO (mini-sMMO), with a turnover frequency of 0.32 s-1, through an optimal reassembly of minimal and modified components of sMMO on catalytically inert and stable apoferritin scaffold. We characterise the molecular characteristics in detail through in silico and experimental analyses and verifications. Notably, in-situ methanol production in a high-cell-density culture of mini-sMMO-expressing recombinant Escherichia coli resulted in higher yield and productivity (~ 3.0 g/L and 0.11 g/L/h, respectively) compared to traditional methanotrophic production.