Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production (original) (raw)
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Applied Biochemistry and Microbiology, 2016
The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3 : 1) as compared to the parental strain (1.1 : 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5 : 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.
Applied Microbiology and Biotechnology, 2020
The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clonebased transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass. Keywords Itaconic acid (IA). Aconitic acid (AA). Aspergillus pseudoterreus ATCC32359. cis-aconitic acid decarboxylase (cadA). Transporter
Bioengineering
Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris, a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris. To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.
Benchmarking recombinant Pichia pastoris for 3‐hydroxypropionic acid production from glycerol
2021
The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3‐hydroxypropionic acid (3‐HP) from glycerol. 3‐HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum‐based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3‐HP from glycerol through the malonyl‐CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14‐fold increase in the production of 3‐HP compared to the initial strain. The best strain (PpHP6) was tested in a fed‐batch culture, achieving a final conc...
Natural Resources for Human Health, 2021
Metabolic engineering is defined as recombinant DNA technology to improve specific biochemical reactions for product formation. We modify the metabolic processes of bacteria to get our desired food by metabolic engineering. Metabolic engineering will enhance these microorganisms' properties and their ability to produce a diverse number of products cost-effectively. To produce amino acids, we modify the central metabolic pathway, biosynthetic pathway, and transport pathway. In many food industries, the production of organic acids through different processes and techniques have proved very beneficial because of their widespread applications. In line with this information, the present review aimed to provide background information for researchers about genetically modified foods for increased food yield to fulfil the nutritional values for average body growth.
Production of Dicarboxylic Acid Platform Chemicals Using Yeasts
Biotransformation of Agricultural Waste and By-Products, 2016
The biotechnological production of biobased dicarboxylic acids has recently become a hot topic in industrial biotechnology, with many investments involved in the development, piloting, and validation at demonstration scale of diverse processes using renewable raw materials. This chapter will review the main markets and applications of commercially relevant dicarboxylic acids and will briefly present their current chemical and biotechnological production processes. The chapter will mainly focus on the particular case of succinic acid. The microbial platforms that have been proposed will be reviewed with emphasis on yeast strains. The basic requirements for setting up and scaling the bioprocess and the required purification strategy to obtain an economically feasible process yielding a product meeting the required specifications will be presented. Throughout the chapter, the specific challenges of using very low-cost raw materials such as agro-industrial residues will be highlighted.
Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts
Applied and environmental microbiology, 1999
Interest in the production of L-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovine L-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into a K. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter(-1), 0.91 g liter(-1) h(-1), and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those...
Metabolic engineering of Pichia pastoris for production of isobutanol and isobutyl acetate
Biotechnology for Biofuels, 2018
Background: Interests in renewable fuels have exploded in recent years as the serious effects of global climate change become apparent. Microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered Pichia pastoris, an industrial workhorse in heterologous enzyme production, to produce the biofuel isobutanol from two renewable carbon sources, glucose and glycerol. Our strategy exploited the yeast's amino acid biosynthetic pathway and diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production. To further demonstrate the versatility of our yeast platform, we incorporated a broad-substrate-range alcohol-O-acyltransferase to generate a variety of volatile esters, including isobutyl acetate ester and isopentyl acetate ester. Results: The engineered strain overexpressing the keto-acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with 2-ketoisovalerate. To improve the production of isobutanol and eliminate the need to supplement the production media with the expensive 2-ketoisovalerate intermediate, we overexpressed a portion of the amino acid l-valine biosynthetic pathway in the engineered strain. While heterologous expression of the pathway genes from the yeast Saccharomyces cerevisiae did not lead to improvement in isobutanol production in the engineered P. pastoris, overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes by employing an episomal plasmid-based expression system further improved the production titer of isobutanol to 2.22 g/L, a 43-fold improvement from the levels observed in the original strain. Finally, heterologous expression of a broad-substraterange alcohol-O-acyltransferase led to the production of isobutyl acetate ester and isopentyl acetate ester at 51 and 24 mg/L, respectively. Conclusions: In this study, we engineered high-level production of the biofuel isobutanol and the corresponding acetate ester by P. pastoris from readily available carbon sources. We envision that our work will provide an economic route to this important class of compounds and establish P. pastoris as a versatile production platform for fuels and chemicals.
Journal of microbiology and biotechnology, 2017
Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant ...