1.Strain engineering and fermentation technology for production of long-chain dicarboxylic acid: a review.
Quan ZHANG ; Zhiqiong WEN ; Lin ZHANG ; Yachao FAN ; Fuli LI
Chinese Journal of Biotechnology 2022;38(12):4420-4431
Long-chain dicarboxylic acid (DCA), a building block for synthesizing a variety of high value-added chemicals, has been widely used in agriculture, chemical, and pharmaceutical industries. The global demand for DCA is increasing in recent years. Compared with chemical synthesis which requires harsh conditions and complicated processes, fermentative production of DCA has many unparalleled advantages, such as low cost and mild reaction conditions. In this review, we summarized the chemical and microbial synthesis methods for DCA and the commercialization status of the fermentation process. Moreover, the advances of using molecular and metabolic engineering to create high-yielding strains for efficient production of DCA were highlighted. Furthermore, the challenges remaining in the microbial fermentation process were also discussed. Finally, the perspectives for developing high titer DCA producing strains by synthetic biology were proposed.
Fermentation
;
Dicarboxylic Acids/metabolism*
;
Metabolic Engineering
;
Technology
2.Recent progress in the biosynthesis of dicarboxylic acids, a monomer of biodegradable plastics.
Rui ZHI ; Yanbo LU ; Min WANG ; Guohui LI ; Yu DENG
Chinese Journal of Biotechnology 2023;39(5):2081-2094
Plastics are one of the most important polymers with huge global demand. However, the downsides of this polymer are that it is difficult to degrade, which causes huge pollution. The environmental-friendly bio-degradable plastics therefore could be an alternative and eventually fulfill the ever-growing demand from every aspect of the society. One of the building blocks of bio-degradable plastics is dicarboxylic acids, which have excellent biodegradability and numerous industrial applications. More importantly, dicarboxylic acid can be biologically synthesized. Herein, this review discusses the recent advance on the biosynthesis routes and metabolic engineering strategies of some of the typical dicarboxylic acids, in hope that it will help to provide inspiration to further efforts on the biosynthesis of dicarboxylic acids.
Biodegradable Plastics
;
Dicarboxylic Acids
;
Polymers/metabolism*
;
Biodegradation, Environmental
;
Metabolic Engineering
3.Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1.
Wen-Na BAO ; Zi-Sheng LUO ; Shi-Wang LIU ; Yuan-Feng WU ; Pei-Lian WEI ; Gong-Nian XIAO ; Yong LIU
Journal of Zhejiang University. Science. B 2019;20(12):995-1002
OBJECTIVE:
This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1.
METHODS:
Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.
RESULTS:
The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °C) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.
CONCLUSIONS
The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.
Alphaproteobacteria/enzymology*
;
Cloning, Molecular
;
Dicarboxylic Acids/metabolism*
;
Enzyme Stability
;
Epoxide Hydrolases/metabolism*
4.Measurement of intracellular pH in long-chain dicarboxylic acid-producing yeast Candida tropicalis and its growth activity.
Shu-Chen LIU ; Lan-Yi XIE ; Chun LI ; Zhu-An CAO
Chinese Journal of Biotechnology 2004;20(2):279-283
Intracellular pH (pHi) has an important influence on the metabolic activity of cells or cellular processes. The intracellular pH (pHi) of long-chain alpha,omega-dicarboxylic acid-producing Candida tropicalis was determined by fluorescence technique using a pH-sensitive fluorescent probe 5(6)-carboxyfluorescein diacetate. Optimal loading conditions of the fluorescent probe into the cells were experimentally determined. Effects of extracellular pH and carbon sources for growth on pHi in the cell grown in a flask were studied; the results indicated that extracellular pH has a slight influence on pHi, whereas carbon sources such as sucrose, glucose, acetic acid, and n-tridecane showed moderate influences. Further work on the relationship between the cell growth activity and pHi was carried out in a 5 L bioreactor. The time course of specific rates of the cell growth, glucose consumption, CO2 production, and pH gradients across cell plasma membrane were plotted, where the cell growth was improved by the higher pHi at 8 - 12 h. The measured pHi values were varied from 5.72 to 6.15 at medium pH 6.0 in which glucose and sodium acetate were used together as carbon source. The investigation of pHi can be helpful for understanding its effects on the kinetics of the metabolic steps involved in the synthesis rate of alpha,omega-dicarboxylic acid and alpha,omega-dicarboxylic acid transport across plasma membranes.
Candida tropicalis
;
growth & development
;
metabolism
;
Culture Media
;
Culture Techniques
;
methods
;
Dicarboxylic Acids
;
metabolism
;
Hydrogen-Ion Concentration
;
Microscopy, Fluorescence
5.Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss.
Kimito HIRAI ; Hisako FURUSHO ; Kiichi HIROTA ; Hajime SASAKI
International Journal of Oral Science 2018;10(2):12-12
Hypoxia (low oxygen level) is an important feature during infections and affects the host defence mechanisms. The host has evolved specific responses to address hypoxia, which are strongly dependent on the activation of hypoxia-inducible factor 1 (HIF-1). Hypoxia interferes degradation of HIF-1 alpha subunit (HIF-1α), leading to stabilisation of HIF-1α, heterodimerization with HIF-1 beta subunit (HIF-1β) and subsequent activation of HIF-1 pathway. Apical periodontitis (periapical lesion) is a consequence of endodontic infection and ultimately results in destruction of tooth-supporting tissue, including alveolar bone. Thus far, the role of HIF-1 in periapical lesions has not been systematically examined. In the present study, we determined the role of HIF-1 in a well-characterised mouse periapical lesion model using two HIF-1α-activating strategies, dimethyloxalylglycine (DMOG) and adenovirus-induced constitutively active HIF-1α (CA-HIF1A). Both DMOG and CA-HIF1A attenuated periapical inflammation and tissue destruction. The attenuation in vivo was associated with downregulation of nuclear factor-κappa B (NF-κB) and osteoclastic gene expressions. These two agents also suppressed NF-κB activation and subsequent production of proinflammatory cytokines by macrophages. Furthermore, activation of HIF-1α by DMOG specifically suppressed lipopolysaccharide-stimulated macrophage differentiation into M1 cells, increasing the ratio of M2 macrophages against M1 cells. Taken together, our data indicated that activation of HIF-1 plays a protective role in the development of apical periodontitis via downregulation of NF-κB, proinflammatory cytokines, M1 macrophages and osteoclastogenesis.
Alveolar Bone Loss
;
metabolism
;
prevention & control
;
Amino Acids, Dicarboxylic
;
pharmacology
;
Animals
;
Cytokines
;
metabolism
;
Down-Regulation
;
Gene Expression
;
drug effects
;
Hypoxia-Inducible Factor 1, alpha Subunit
;
physiology
;
Macrophages
;
physiology
;
Mice
;
NF-kappa B
;
metabolism
;
Osteogenesis
;
physiology
;
Periapical Periodontitis
;
metabolism
;
prevention & control
;
Real-Time Polymerase Chain Reaction
;
X-Ray Microtomography