Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy
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ABSTRACT:
SUBMITTER: Zeng W
PROVIDER: S-EPMC10992570 | biostudies-literature | 2022 Nov
REPOSITORIES: biostudies-literature
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Project description:Background(R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains.ResultsHerein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate.ConclusionsThe microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.
| S-EPMC9838050 | biostudies-literature
Project description:Background2,3-Butanediol is a platform and fuel biochemical that can be efficiently produced from biomass. However, a value-added process for this chemical has not yet been developed. To expand the utilization of 2,3-butanediol produced from biomass, an improved derivative process of 2,3-butanediol is desirable.ResultsIn this study, a Gluconobacter oxydans strain DSM 2003 was found to have the ability to transform 2,3-butanediol into acetoin, a high value feedstock that can be widely used in dairy and cosmetic products, and chemical synthesis. All three stereoisomers, meso-2,3-butanediol, (2R,3R)-2,3-butanediol, and (2S,3S)-2,3-butanediol, could be transformed into acetoin by the strain. After optimization of the bioconversion conditions, the optimum growth temperature for acetoin production by strain DSM 2003 was found to be 30°C and the medium pH was 6.0. With an initial 2,3-butanediol concentration of 40 g/L, acetoin at a high concentration of 89.2 g/L was obtained from 2,3-butanediol by fed-batch bioconversion with a high productivity (1.24 g/L · h) and high yield (0.912 mol/mol).ConclusionsG. oxydans DSM 2003 is the first strain that can be used in the direct production of acetoin from 2,3-butanediol. The product concentration and yield of the novel process are both new records for acetoin production. The results demonstrate that the method developed in this study could provide a promising process for efficient acetoin production and industrially produced 2,3-butanediol utilization.
| S-EPMC4177140 | biostudies-literature
Project description:Acetic acid bacteria are well-known for their ability to incompletely oxidize their carbon sources. Many of the products of these oxidations find industrial uses. Metabolic engineering of acetic acid bacteria would improve production efficiency and yield by allowing controllable gene expression. However, the molecular tools necessary for regulating gene expression have only recently started being explored. To this end the ability of the activation-dependent Plux system and two constitutive repression Ptet systems were examined for their ability to modulate gene expression in Gluconobacter oxydans. The activation-dependent Plux system increased gene expression approximately 5-fold regardless of the strength of the constitutive promoter used to express the luxR transcriptional activator. The Ptet system was tunable and had a nearly 20-fold induction when the tetR gene was expressed from the strong constitutive promoters P0169 and P264, but only had a 4-fold induction when a weak constitutive promoter (P452) was used for tetR expression. However, the Ptet system was somewhat leaky when uninduced. To mitigate this background activity, a bicistronic TetR expression system was constructed. Based on molecular modeling, this system is predicted to have low background activity when not induced with anhydrotetracycline. The bicistronic system was inducible up to >3,000-fold and was highly tunable with almost no background expression when uninduced, making this bicistronic system potentially useful for engineering G. oxydans and possibly other acetic acid bacteria. These expression systems add to the newly growing repertoire of suitable regulatable promoter systems in acetic acid bacteria.
| S-EPMC9306550 | biostudies-literature
Project description:BackgroundHeme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme.ResultsThe heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter.ConclusionsStrengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.
| S-EPMC10193802 | biostudies-literature
Project description:BackgroundThe global market for sweeteners is increasing, and the food industry is constantly looking for new low-caloric sweeteners. The natural sweetener 5-keto-D-fructose is one such candidate. 5-Keto-D-fructose has a similar sweet taste quality as fructose. Developing a highly efficient 5-keto-D-fructose production process is key to being competitive with established sweeteners. Hence, the 5-keto-D-fructose production process was optimised regarding titre, yield, and productivity.ResultsFor production of 5-keto-D-fructose with G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST an extended-batch fermentation was conducted. During fructose feeding, a decreasing respiratory activity occurred, despite sufficient carbon supply. Oxygen and second substrate limitation could be excluded as reasons for the decreasing respiration. It was demonstrated that a short period of oxygen limitation has no significant influence on 5-keto-D-fructose production, showing the robustness of this process. Increasing the medium concentration increased initial biomass formation. Applying a fructose feeding solution with a concentration of approx. 1200 g/L, a titre of 545 g/L 5-keto-D-fructose was reached. The yield was with 0.98 g5-keto-d-fructose/gfructose close to the theoretical maximum. A 1200 g/L fructose solution has a viscosity of 450 mPa∙s at a temperature of 55 °C. Hence, the solution itself and the whole peripheral feeding system need to be heated, to apply such a highly concentrated feeding solution. Thermal treatment of highly concentrated fructose solutions led to the formation of 5-hydroxymethylfurfural, which inhibited the 5-keto-D-fructose production. Therefore, fructose solutions were only heated to about 100 °C for approx. 10 min. An alternative feeding strategy was investigated using solid fructose cubes, reaching the highest productivities above 10 g5-keto-d-fructose/L/h during feeding. Moreover, the scale-up of the 5-keto-D-fructose production to a 150 L pressurised fermenter was successfully demonstrated using liquid fructose solutions (745 g/L).ConclusionWe optimised the 5-keto-D-fructose production process and successfully increased titre, yield and productivity. By using solid fructose, we presented a second feeding strategy, which can be of great interest for further scale-up experiments. A first scale-up of this process was performed, showing the possibility for an industrial production of 5-keto-D-fructose.
| S-EPMC9741787 | biostudies-literature
Project description:l-Sorbose is an essential intermediate for the industrial production of vitamin C (l-ascorbic acid). However, the formation of fructose and some unknown by-products significantly reduces the conversion ratio of D-sorbitol to l-sorbose. This study aimed to identify the key D-sorbitol dehydrogenases in Gluconobacter oxydans WSH-003 by gene knockout. Then, a total of 38 dehydrogenases were knocked out in G. oxydans WSH-003, and 23 dehydrogenase-deficient strains could increase l-sorbose production. G. oxydans-30, wherein a pyrroloquinoline quinone-dependent glucose dehydrogenase was deleted, showed a significant reduction of a by-product with the extension of fermentation time. In addition, the highest conversion ratio of 99.60% was achieved in G. oxydans MD-16, in which 16 different types of dehydrogenases were inactivated consecutively. Finally, the gene vhb encoding hemoglobin was introduced into the strain. The titer of l-sorbose was 298.61 g/L in a 5-L bioreactor. The results showed that the systematic engineering of dehydrogenase could significantly enhance the production of l-sorbose.
| S-EPMC8927921 | biostudies-literature
Project description:Polysaccharides are important natural biomacromolecules. In particular, microbial exopolysaccharides have received much attention. They are produced by a variety of microorganisms, and they are widely used in the food, pharmaceutical, and chemical industries. The Candida glabrata mutant 4-C10, which has the capacity to produce exopolysaccharide, was previously obtained by random mutagenesis. In this study we aimed to further enhance exopolysaccharide production by systemic fermentation optimization. By single factor optimization and orthogonal design optimization in shaking flasks, an optimal fermentation medium composition was obtained. By optimizing agitation speed, aeration rate, and fed-batch fermentation mode, 118.6 g L-1 of exopolysaccharide was obtained by a constant rate feeding fermentation mode, with a glucose yield of 0.62 g g-1 and a productivity of 1.24 g L-1 h-1. Scaling up the established fermentation mode to a 15-L fermenter led to an exopolysaccharide yield of 113.8 g L-1, with a glucose yield of 0.60 g g-1 and a productivity of 1.29 g L-1 h-1.
| S-EPMC9478339 | biostudies-literature
Project description:BackgroundWhole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications.ResultsG. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ?GOX1068?GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ?GOX1068?GOX0854 as the biocatalyst, 61.6 g L-1 xylonate was produced from 58.4 g L-1 xylose at a yield of 1.05 g g-1.ConclusionThis process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol.
| S-EPMC6174558 | biostudies-literature
Project description:BackgroundL-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated.ResultsAn efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h.ConclusionsThe 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme and respiratory chain engineering. Fed-batch fermentation could further enlarge the positive effect and increase the 5-KGA production. All of these demonstrated that the robust recombinant strain can efficiently produce 5-KGA in larger scale to fulfill the industrial production of L-TA from 5-KGA.
| S-EPMC4869267 | biostudies-literature
Project description:BackgroundThe acetic acid bacterium Gluconobacter oxydans 621H is characterized by its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. The metabolism of this α-proteobacterium has been characterized to some extent, yet little is known about its transcriptomes and related data. In this study, we applied two different RNAseq approaches. Primary transcriptomes enriched for 5'-ends of transcripts were sequenced to detect transcription start sites, which allow subsequent analysis of promoter motifs, ribosome binding sites, and 5´-UTRs. Whole transcriptomes were sequenced to identify expressed genes and operon structures.ResultsSequencing of primary transcriptomes of G. oxydans revealed 2449 TSSs, which were classified according to their genomic context followed by identification of promoter and ribosome binding site motifs, analysis of 5´-UTRs including validation of predicted cis-regulatory elements and correction of start codons. 1144 (41%) of all genes were found to be expressed monocistronically, whereas 1634 genes were organized in 571 operons. Together, TSSs and whole transcriptome data were also used to identify novel intergenic (18), intragenic (328), and antisense transcripts (313).ConclusionsThis study provides deep insights into the transcriptional landscapes of G. oxydans. The comprehensive transcriptome data, which we made publicly available, facilitate further analysis of promoters and other regulatory elements. This will support future approaches for rational strain development and targeted gene expression in G. oxydans. The corrections of start codons further improve the high quality genome reference and support future proteome analysis.
| S-EPMC5756330 | biostudies-literature