scholarly journals Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus

2021 ◽  
Vol 9 ◽  
Author(s):  
Kyle R. Pomraning ◽  
Ziyu Dai ◽  
Nathalie Munoz ◽  
Young-Mo Kim ◽  
Yuqian Gao ◽  
...  
Keyword(s):  
Organic Acids ◽  
Filamentous Fungus ◽  
Shake Flask ◽  
Metabolic Flux ◽  
High Titer ◽  
Value Added ◽  
Model Systems ◽  
Shake Flask Culture ◽  
Semialdehyde Dehydrogenase ◽  
Hydroxypropionic Acid

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

scholarly journals Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

2006 ◽  
Vol 73 (3) ◽  
pp. 718-729 ◽  
Author(s):  
Yinjie J. Tang ◽  
Judy S. Hwang ◽  
David E. Wemmer ◽  
Jay D. Keasling
Keyword(s):  
Metabolic Pathway ◽  
Uptake Rate ◽  
Shake Flask ◽  
Metabolic Flux ◽  
Environmental Changes ◽  
Shewanella Oneidensis ◽  
Tca Cycle ◽  
Shake Flask Culture ◽  
Flux Analysis ◽  
Lactate Uptake

ABSTRACT The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

scholarly journals Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chaofeng Li ◽  
Xiaofeng Lin ◽  
Xing Ling ◽  
Shuo Li ◽  
Hao Fang
Keyword(s):  
Acid Production ◽  
Microbial Consortium ◽  
Consolidated Bioprocessing ◽  
High Titer ◽  
Value Added ◽  
Superior Performance ◽  
Glucaric Acid ◽  
Practical Applications ◽  
Work Distribution ◽  
Rut C30

Abstract Background The biomanufacturing of d-glucaric acid has attracted increasing interest because it is one of the top value-added chemicals produced from biomass. Saccharomyces cerevisiae is regarded as an excellent host for d-glucaric acid production. Results The opi1 gene was knocked out because of its negative regulation on myo-inositol synthesis, which is the limiting step of d-glucaric acid production by S. cerevisiae. We then constructed the biosynthesis pathway of d-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA-C, producing record-breaking titers of d-glucaric acid: 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L d-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in fed-batch fermentation mode, respectively. However, LGA-1 was preferable because of its genetic stability and its superior performance in practical applications. There have been no reports on d-glucaric acid production from lignocellulose. Therefore, the biorefinery processes, including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated and compared. CBP using an artificial microbial consortium composed of Trichoderma reesei (T. reesei) Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high d-glucaric acid titers and yields after 7 d of fermentation, 0.54 ± 0.12 g/L d-glucaric acid from 15 g/L Avicel and 0.45 ± 0.06 g/L d-glucaric acid from 15 g/L steam-exploded corn stover (SECS), respectively. In an attempt to design the microbial consortium for more efficient CBP, the team consisting of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be the best, with excellent work distribution and collaboration. Conclusions Two engineered S. cerevisiae strains, LGA-1 and LGA-C, with high titers of d-glucaric acid were obtained. This indicated that S. cerevisiae INVSc1 is an excellent host for d-glucaric acid production. Lignocellulose is a preferable substrate over myo-inositol. SHF, SSF, and CBP were studied, and CBP using an artificial microbial consortium of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be promising because of its relatively high titer and yield. T. reesei Rut-C30 and S. cerevisiae LGA-1were proven to be the best teammates for CBP. Further work should be done to improve the efficiency of this microbial consortium for d-glucaric acid production from lignocellulose.

Solubility of Iron in Model Systems Containing Organic Acids and Lignin

1992 ◽  
Vol 55 (11) ◽  
pp. 893-898 ◽  
Author(s):  
TAKESHI SUZUKI ◽  
FERGUS M. CLYDESDALE ◽  
TIRA PANDOLF
Keyword(s):  
Ascorbic Acid ◽  
Organic Acids ◽  
Model Systems ◽  
Molar Ratio ◽  
Soluble Form ◽  
Enhancing Effect ◽  
Gastrointestinal Ph ◽  
Soluble Iron ◽  
Ph Values ◽  
Ph Conditions

The effect of six organic acids, ascorbic, citric, fumaric, lactic, malic, and succinic, alone and in combination, at a 1:1.9 molar ratio (Fe+2:ligand) on the solubility of iron was evaluated in the presence of lignin under simulated gastrointestinal pH conditions. The enhancing effect, evaluated under two systems of preparation at two pH values, was in the following order: citric>malic>ascorbic>lactic,fumaric>succinic. Citric acid solubilized 80 and 81% of iron under both pH conditions. When ascorbic acid was mixed with fumaric, lactic, and succinic acids, a higher percentage of soluble iron was retained than with these three acids alone. In the case of citric and malic acids, the addition of ascorbic acid reduced the soluble iron. The percentage of soluble iron obtained when prepared at the endogenous pH (2.5–3.1) was higher than that at pH 5.5. These results indicated that ascorbate bound less iron in a soluble form than citrate or malate but more than fumarate, lactate, or succinate. Also, combinations of citric with malic acid did not demonstrate a synergistic effect.

scholarly journals Optimization of Medium Ingredients for Keratinolytic Protease Production by Bacillus licheniformis MZK-03 using Statistical Experimental Designs

1970 ◽  
Vol 24 (1) ◽  
pp. 52-56 ◽  
Author(s):  
Mohammad Moniruzzaman ◽  
Alamgir Rahman ◽  
M Mozammel Hoq
Keyword(s):  
Trace Elements ◽  
Bacillus Licheniformis ◽  
Shake Flask ◽  
Culture Medium ◽  
Protease Production ◽  
Shake Flask Culture ◽  
Flask Culture ◽  
Statistical Approaches ◽  
Central Composite ◽  
Keratinolytic Protease

A culture medium was optimized for the production of keratinolytic protease by a newly isolated strain of Bacillus licheniformis MZK-03 in shake-flask culture. Based on the results of preliminary experiments, feather mill, molasses and trace elements were found to be major variables in keratinolytic protease production. The concentrations of these ingredients were optimized by using two statistical approaches, namely Box-Wilson method and central composite design. The optimized culture medium, finally determined by using the statistical approaches, composed of 0.95% feather mill, 0.12% molasses and 1.44% trace elements. The keratinolytic protease production was increased by approximately 2-fold when the strain was grown in the optimized medium (95.2 U/ml) compared to the un-optimized medium (56.05 U/ml). Keywords: Keratinolytic protease, Optimization, Bacillus licheniformis MZK-03, Statistical designsDOI: http://dx.doi.org/10.3329/bjm.v24i1.1238 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 52-56

scholarly journals Engineering Corynebacterium glutamicum for the Efficient Production of 3-Hydroxypropionic Acid from a Mixture of Glucose and Acetate via the Malonyl-CoA Pathway

Catalysts ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 203
Author(s):  
Zhishuai Chang ◽  
Wei Dai ◽  
Yufeng Mao ◽  
Zhenzhen Cui ◽  
Zhiwen Wang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Value Added ◽  
Acid Synthesis ◽  
Efficient Production ◽  
Endogenous Gene ◽  
Malonyl Coa ◽  
Acetate Accumulation ◽  
Level I ◽  
Coa Reductase ◽  
Hydroxypropionic Acid

3-Hydroxypropionic acid (3-HP) has been recognized as one of the top value-added building block chemicals, due to its numerous potential applications. Over the past decade, biosynthesis of 3-HP via the malonyl-CoA pathway has been increasingly favored because it is balanced in terms of ATP and reducing equivalents, does not require the addition of costly coenzymes, and can utilize renewable lignocellulosic biomass. In this study, gene mcr encoding malonyl-CoA reductase from Chloroflexus aurantiacus was introduced into Corynebacterium glutamicum ATCC13032 to construct the strain Cgz1, which accumulated 0.30 g/L 3-HP. Gene ldhA encoding lactate dehydrogenase was subsequently deleted to eliminate lactate accumulation, but this decreased 3-HP production and greatly increased acetate accumulation. Then, different acetate utilization genes were overexpressed to reuse the acetate, and the best candidate Cgz5 expressing endogenous gene pta could effectively reduce the acetate accumulation and produced 0.68 g/L 3-HP. To enhance the supply of the precursor acetyl-CoA, acetate was used as an ancillary carbon source to improve the 3-HP production, and 1.33 g/L 3-HP could be produced from a mixture of glucose and acetate, with a 2.06-fold higher yield than from glucose alone. Finally, to inhibit the major 3-HP competing pathway-fatty acid synthesis, 10 μM cerulenin was added and strain Cgz5 produced 3.77 g/L 3-HP from 15.47 g/L glucose and 4.68 g/L acetate with a yield of 187 mg/g substrate in 48 h, which was 12.57-fold higher than that of Cgz1. To our best knowledge, this is the first report on engineering C. glutamicum to produce 3-HP via the malonyl-CoA pathway. The results indicate that the innocuous biosafety level I microorganism C. glutamicum is a potential industrial 3-HP producer.

Differential inhibitory effects of organic acids on pear polyphenol oxidase in model systems and pear puree

LWT ◽  
2020 ◽  
Vol 118 ◽  
pp. 108704 ◽  
Author(s):  
Tao Liao ◽  
Junping Liu ◽  
Yuefang Sun ◽  
Liqiang Zou ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Organic Acids ◽  
Polyphenol Oxidase ◽  
Model Systems ◽  
Inhibitory Effects

scholarly journals Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jun Ren ◽  
Hyang-Mi Lee ◽  
Thi Duc Thai ◽  
Dokyun Na
Keyword(s):  
Genetic Engineering ◽  
Plasmid Dna ◽  
Metabolic Flux ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Value Added ◽  
Cytosine Methyltransferase ◽  
Genome Bisulfite Sequencing ◽  
First Time ◽  
Value Added Products

Abstract Background Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

Study on Screening of Glutathione over-Production Strain and its Fermentation Conditions

2011 ◽  
Vol 396-398 ◽  
pp. 1657-1661
Author(s):  
Jie Gang ◽  
Li Feng Shan ◽  
Hong Yu Bu
Keyword(s):  
Shake Flask ◽  
Fermentation Medium ◽  
Culture Conditions ◽  
Shake Flask Culture ◽  
Optimal Conditions ◽  
Flask Culture ◽  
Fermentation Conditions ◽  
Physiological And Biochemical Characteristics ◽  
Physiological And Biochemical ◽  
Production Strain

This study was to screen strains for glutathione over-production. A high GSH-producing strain HSJB1 was isolated from soil.According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. The glutathion fermentation conditions of this strain by shake flask culture were studied.The optimal fermentation medium was as follows: glucose 20g/L, (NH4)2SO4 5g/L, peptone 12.5 g/L, KH2PO4 9.0 g/L, MgSO4•7H2O 1.0 g/L, NaCl 0.2 g/L, ZnSO4•H2O 0.01 g/L. The optimal culture conditions were as follows :pH 5，broth quantity 30ml/250ml, temperature 28°C，inoculum 10%. Under the optimal conditions, the glutathione productivity and biomass were 53.60 mg/L and 3.8 g/L, respectively, which were 11.2% and 11.8% higher than those in the original conditions, respectively.

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