Evaluation and application of constitutive promoters for cutinase production by Saccharomyces cerevisiae

Juan Zhang,Yanqiu Cai,Guocheng Du,Jian Chen,Miao Wang,Zhen Kang 한국미생물학회 2017 The journal of microbiology Vol.55 No.7

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.

Production of Cellulases by Rhizopus stolonifer from Glucose-Containing Media Based on the Regulation of Transcriptional Regulator CRE

( Yingying Zhang ),( Bin Tang ),( Guocheng Du ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.3

Carbon catabolite repression is a crucial regulation mechanism in microorganisms, but its characteristic in Rhizopus is still unclear. We extracted a carbon regulation gene, cre, that encoded a carbon catabolite repressor protein (CRE) from Rhizopus stolonifer TP-02, and studied the regulation of CRE by real-time qPCR. CRE responded to glucose in a certain range, where it could significantly regulate part of the cellulase genes (eg, bg, and cbh2) without cbh1. In the comparison of the response of cre and four cellulase genes to carboxymethylcellulose sodium and a simple carbon source (lactose), the effect of CRE was only related to the concentration of reducing sugars. By regulating the reducing sugars to range from 0.4% to 0.6%, a glucose-containing medium with lactose as the inducer could effectively induce cellulases without the repression of CRE. This regulation method could potentially reduce the cost of enzymes produced in industries and provide a possible solution to achieve the largescale synthesis of cellulases.

Enhanced Acid Tolerance in Lactobacillus casei by Adaptive Evolution and Compared Stress Response during Acid Stress

Juan Zhang,Chongde Wu,Guocheng Du,Jian Chen 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.2

This study aimed to improve the acid tolerance of Lactobacillus casei Zhang and compare the stress response of the parental strain and the acid-resistant mutant during acidic conditions. Adaptive evolution was conducted for 70 days to generate acid-tolerant L. casei. The evolved mutant lb-2 exhibited more than a 60% increase in biomass as well as a 13.6 and 65.6% increase in concentrations of lactate and acetate, respectively, when cultured at pH 4.3 for 64 h. Lactic acid tolerances of the parental strain and the evolved mutant were determined. As a result,the evolved mutant showed a 318-fold higher survival rate than that of the parental strain. Physiological analysis showed that the evolved mutant exhibited higher intracellular pH (pHi), NH4+ concentration and lower inner membrane permeability than that of the parental strain during acid stress. Moreover, higher amounts of intracellular arginine and aspartate were also detected in lb-2under acid stress. Validation of the relationship between the acid tolerance and the intracellular arginine and aspartate accumulation was conducted by experiments that showed the survival of L. casei at pH 3.3 was improved 1.36-,2.10-, or 3.42-fold by the addition of 50 mM aspartate,arginine or both of them, respectively. Taken together,results presented here not only supply an effective way to select acid-resistant strains for the food industry, but also contribute to reveal the mechanisms of acid tolerance and provide new strategies to enhance the industrial utility and health-promoting properties of this species.

Distributed control system architecture for deep submergence rescue vehicles

Yushan Sun,Xiangrui Ran,Guocheng Zhang,Fanyu Wu,Chengrong Du 대한조선학회 2019 International Journal of Naval Architecture and Oc Vol.11 No.1

The control architectures of Chuan Suo (CS) deep submergence rescue vehicle are introduced. The hardware and software architectures are also discussed. The hardware part adopts a distributed control system composed of surface and underwater nodes. A computer is used as a surface control machine. Underwater equipment is based on a multi-board-embedded industrial computer with PC104 BUS, which contains IO, A/D, D/A, eight-channel serial, and power boards. The hardware and software parts complete data transmission through optical fibers. The software part involves an IPC of embedded Vxworks real-time operating system, upon which the operation of I/O, A/D, and D/A boards and serial ports is based on; this setup improves the real-time manipulation. The information flow is controlled by the software part, and the thrust distribution is introduced. A submergence vehicle heeling control method based on ballast water tank regulation is introduced to meet the special heeling requirements of the submergence rescue vehicle during docking. Finally, the feasibility and reliability of the entire system are verified by a pool test.

Screening and Characterization of an Aerobic Nitrifying-denitrifying Bacterium from Activated Sludge

Xiaofan Qiu,Tianwen Wang,Xiaomin Zhong,Guocheng Du,Jian Chen 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.2

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes,nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH4+-N and NO3−-N. When the initial concentrations of NH4+-N and NO3−-N in synthetic wastewater were less than 70 and 50mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.

Distributed control system architecture for deep submergence rescue vehicles

Sun, Yushan,Ran, Xiangrui,Zhang, Guocheng,Wu, Fanyu,Du, Chengrong The Society of Naval Architects of Korea 2019 International Journal of Naval Architecture and Oc Vol.11 No.1

The control architectures of Chuan Suo (CS) deep submergence rescue vehicle are introduced. The hardware and software architectures are also discussed. The hardware part adopts a distributed control system composed of surface and underwater nodes. A computer is used as a surface control machine. Underwater equipment is based on a multi-board-embedded industrial computer with PC104 BUS, which contains IO, A/D, D/A, eight-channel serial, and power boards. The hardware and software parts complete data transmission through optical fibers. The software part involves an IPC of embedded Vxworks real-time operating system, upon which the operation of I/O, A/D, and D/A boards and serial ports is based on; this setup improves the real-time manipulation. The information flow is controlled by the software part, and the thrust distribution is introduced. A submergence vehicle heeling control method based on ballast water tank regulation is introduced to meet the special heeling requirements of the submergence rescue vehicle during docking. Finally, the feasibility and reliability of the entire system are verified by a pool test.

Combinatorial Fine-Tuning of Phospholipase D Expression by Bacillus subtilis WB600 for the Production of Phosphatidylserine

( Tingting Huang ),( Xueqin Lv ),( Jianghua Li ),( Hyun-dong Shin ),( Guocheng Du ),( Long Liu ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.

Biocatalytic Production of Glucosamine from N-Acetylglucosamine by Diacetylchitobiose Deacetylase

( Zhu Jiang ),( Xueqin Lv ),( Yanfeng Liu ),( Hyun-dong Shin ),( Jianghua Li ),( Guocheng Du ),( Long Liu ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.11

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dac_ph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for largescale production of GlcN in an environment-friendly manner.

Engineering of Biosynthesis Pathway and NADPH Supply for Improved L-5-Methyltetrahydrofolate Production by Lactococcus lactis

( Chuanchuan Lu ),( Yanfeng Liu ),( Jianghua Li ),( Long Liu ),( Guocheng Du ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.1

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 μg/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 μg/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5- MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 μg/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 μg/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 μg/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

Enhanced Cutinase Production of Thermobifida fusca by a Two-stage Batch and Fed-batch Cultivation Strategy

Gangqiang He,Guanghua Huo,Liming Liu,Yang Zhu,Jian Chen,Guocheng Du 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.1

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11~20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and con-sumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.