Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose

( Xinyao Lu ),( Xiaomeng Fu ),( Hong Zong ),( Bin Zhuge ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.6

1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD+ ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae.

The N-Terminal α-Helix Domain of Pseudomonas aeruginosa Lipoxygenase Is Required for Its Soluble Expression in Escherichia coli but Not for Catalysis

( Xinyao Lu ),( Guangsheng Wang ),( Yue Feng ),( Song Liu ),( Xiaoman Zhou ),( Guocheng Du ),( Jian Chen ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.10

Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure of Pseudomonas aeruginosa LOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this Nterminal helix domain in the soluble expression and catalysis of P. aeruginosa LOX were analyzed. Genetic truncation of this helix domain resulted in an insoluble P. aeruginosa LOX mutant. The active C-terminal domain was obtained by dispase digestion of the P. aeruginosa LOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain of P. aeruginosa LOX is required for its soluble expression in E. coli, which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain of P. aeruginosa LOX and further improvement of its enzyme properties by molecular modification.

Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose

Lu, Xinyao,Fu, Xiaomeng,Zong, Hong,Zhuge, Bin The Korean Society for Microbiology and Biotechnol 2016 Journal of microbiology and biotechnology Vol.26 No.7

1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD⁺ ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae.

Bioproduction of trans-10,cis-12-Conjugated Linoleic Acid by a Highly Soluble and Conveniently Extracted Linoleic Acid Isomerase and an Extracellularly Expressed Lipase from Recombinant Escherichia coli Strains

( Mengnan Huang ),( Xinyao Lu ),( Hong Zong ),( Bin Zhuge ),( Wei Shen ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.5

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJGrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJGrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

Effect of Goji Berry extract on cell viability of Lactiplantibacillus plantarum M5 microcapsules during in vitro gastrointestinal digestion

Jingrui Zheng,Yiqi Li,Xinyao Lu,Bin Zhuge,Hong Zong 한국식품과학회 2024 Food Science and Biotechnology Vol.33 No.8

Lactiplantibacillus plantarum M5 and Goji Berry extract were co-microencapsulated to maintain the activity of cells during gastrointestinal digestion, and the mechanism by which they could maintain high activity was investigated. The results showed that the microcapsules with 3% Goji Berry extract(A-GE-3) had the largest encapsulation efficiency(EE) of 92.41 ± 0.58%. SEM showed that the structure of A-GE-3 microcapsules were smoother and denser. Cell viability in A-GE-3 microcapsules remained at 7.17 log10 CFU/g after gastrointestinal digestion. Meanwhile, during the gastrointestinal digestion with 3% Goji Berry extract, cell membrane damage detected by fluorescent probes propidium iodide(PI) and 1.1-N-phenylnaphthylamine(NPN) was significantly reduced; the contents of arginine, glutamic acid and oleic acid in cell membrane were increased, which helped to maintain the dynamic balance of intracellular pH and regulated cell membrane fluidity in response to gastrointestinal environment. This study demonstrated the potential of Goji Berry extract as a probiotic protector in gastrointestinal digestion.

Synthesis of pinene in the industrial strain Candida glycerinogenes by modification of its mevalonate pathway

Ma Tengfei,Zong Hong,Lu Xinyao,Zhuge Bin 한국미생물학회 2022 The journal of microbiology Vol.60 No.12

Terpenes have many applications and are widely found in nature, but recent progress in synthetic biology has enabled the use of microorganisms as chassis cells for the synthesis of these compounds. Candida glycerinogenes (C. glycerinogenes) is an industrial strain that may be developed as a chassis for the synthesis of terpenes since it has a tolerance to hyperosmolality and high sugar, and has a complete mevalonate (MVA) pathway. However, monoterpenes such as pinene are highly toxic, and the tolerance of C. glycerinogenes to pinene was investigated. We also measured the content of mevalonate and squalene to evaluate the strength of the MVA pathway. To determine terpene synthesis capacity, a pathway for the synthesis of pinene was constructed in C. glycerinogenes. Pinene production was improved by overexpression, gene knockdown and antisense RNA inhibition. Pinene production was mainly enhanced by strengthening the upstream MVA pathway and inhibiting the production of by-products from the downstream pathway. With these strategies, yield could be increased by almost 16 times, to 6.0 mg/L. Overall, we successfully constructed a pinene synthesis pathway in C. glycerinogenes and enhanced pinene production through metabolic modification.

Immobilization of Acetobacter sp. CGMCC 8142 for Efficient Biocatalysis of 1, 3-propanediol to 3-hydroxypropionic Acid

Jun Li,Hong Zong,Bin Zhuge,Xinyao Lu,Huiying Fang,Jin Sun 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.4

3-hydroxypropionic acid (3-HP) is an important chemical platform organic in material industry, daily chemical industry and biomedicine field due to its numerous valuable derivatives. However, no mature methods have been established in the synthesis industry for direct large scale production. The bacterium Acetobacter sp. CGMCC 8142 with high efficiency of alcohols oxidation property was immobilized for biosynthesis of 3-HP from 1, 3- propanediol (1, 3-PDO). Parameter values in mass transfer modeling indicated that mass transfer of immobilized biocatalysts affected the oxidation reaction (the internal effectiveness factor ηi < 1) but was not the rate-limiting step if Thiele modulus 1 > ϕ > 0.3. The optimal immobilization conditions for 3-HP biocatalysis was sodium alginate 40 g/L, gel beads diameter 1 mm, cross-linkage time 2 h and 0.1mM FeCl2. Immobilized cells showed promising substrate tolerance, pH stability, thermal stability and storability. After 5 cycles of reaction, 3-HP molar yield of immobilized beads was retained to 80.26%, and 66.95 g/L 3-HP were produced from 70 g/L 1, 3-PDO. The biocatalysis process of immobilized cells introduced in this study may provide an economical and efficiency alternative route for practical production of 3-HP.

Protoplast Preparation and Polyethylene Glycol (PEG)-mediated Transformation of Candida glycerinogenes

Cheng Zhang,Hong Zong,Bin Zhuge,Xinyao Lu,Huiying Fang,Jiali Zhu,Jian Zhuge 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.1

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.