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1.
Jiwei Mao Quanli Liu Xiaofei Song Hesuiyuan Wang Hui Feng Haijin Xu Mingqiang Qiao 《Biotechnology letters》2017,39(7):977-982
Objective
To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.Result
When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.Conclusion
Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.2.
Objective
To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.Results
Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l?1, 100 g wet cells l?1, and 25 g LA l?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l?1.Conclusion
Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.3.
Shu Zhang Jian Huang Rong Hu Guo Guo Xiaoli Shang Jianwei Wu 《Biotechnology letters》2017,39(8):1219-1227
Objective
To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications.Results
A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l?1. The enzyme has the maximum activity with 27.6 U mg?1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg?1), cellobiose (40.1 ± 2.3 U mg?1) and cellulose (27.6 ± 3.5 U mg?1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites.Conclusions
Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.4.
Objectives
To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.Results
The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K m of 0.15 mM and V max of 62 μmol min?1 mg?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.Conclusions
LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.5.
Deqiang Zhu Jianrong Wu Xiaobei Zhan Li Zhu Zhiyong Zheng Minjie Gao 《Biotechnology letters》2017,39(2):227-234
Objectives
N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).Results
PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l?1, compared with 3.6 ± 0.15 g l?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l?1 in a 1 l fermenter.Conclusions
The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.6.
Wenwen Zhang Zhaohui Chen Mengmeng Wu Zhong Shi Feng Zhu Guoqiang li Ting Ma 《Biotechnology letters》2016,38(6):991-997
Objective
To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.Results
The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l?1 compared to 21 g ± 0.4 g l?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.Conclusions
Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.7.
Van Dung Pham Sivachandiran Somasundaram Seung Hwan Lee Si Jae Park Soon Ho Hong 《Biotechnology letters》2016,38(2):321-327
Objectives
To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli.Results
Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l?1 was produced from 10 g glucose l?1 while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l?1 when 20 g glucose l?1 was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l?1).Conclusions
The novel GABA production system was constructed by co-localization of GABA shunt enzymes.8.
Background
Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.Results
The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l-1 ethanol (0.58 g l-1 h-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l-1 h-1).Conclusions
In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.9.
Tatyana Laurinavichene Kestutis Laurinavichius Evgeny Shastik Anatoly Tsygankov 《Biotechnology letters》2018,40(2):309-314
Objectives
To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.Results
Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l?1 and 0.04 g yeast extract l?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H2 yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.Conclusion
The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H2 production without accumulation of organic acids under conditions of Clostridia limitation.10.
Xiuling Shang Xin Chai Xuemei Lu Yuan Li Yun Zhang Guoqiang Wang Chen Zhang Shuwen Liu Yu Zhang Jiyin Ma Tingyi Wen 《Biotechnology letters》2018,40(2):383-391
Objective
To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering.Results
Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using β-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R 2 = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction.Conclusions
The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.11.
Objectives
To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae.Results
The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4 × 105 CFU μg?1 DNA, which is 10 times higher than that of the wild strain. 52.2 g 1,3-propanediol L?1, 30.7 g 2,3-butanediol L?1, and 175.9 g 2-ketogluconic acid L?1 were produced by non-capsulated mutants, which were 10–40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8–2.2 cP.Conclusions
Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.12.
Objectives
l-isoleucine dioxygenase (IDO) specifically transforms l-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.Results
The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with IdoN126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with IdoT130K showing a 170% increase in activity. Although the activity of the combined mutant IdoN126H/T130K (0.95?±?0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing IdoN126H/T130K and Ido, 66.50?±?0.99 mM and 26.09?±?1.85 mM 4-HIL was synthesized, respectively, in 24 h.Conclusion
IdoN126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.13.
Dashuai Li Qiang Zhang Zhijiang Zhou Fanglong Zhao Wenyu Lu 《Biotechnology letters》2016,38(4):603-609
Objectives
To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.Results
By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l?1 in E. coli under the shake-flask conditions.Conclusions
Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.14.
Objectives
To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production.Results
The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l?1. Both strains reached similar cell densities ranging from 5 to 8.8 g l?1 under the different conditions. The highest pDNA yields on biomass (YpDNA/X) for both strains were obtained when 3 U enzyme l?1were used. Under these conditions, 35 ± 3 mgof pDNA l?1 were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl?1 after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains.Conclusions
The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.15.
Lei Chen Xiaoming Yi Fajun Deng Wei Fang Xuecheng Zhang Xiaotang Wang Zemin Fang Yazhong Xiao 《Biotechnology letters》2016,38(3):471-476
Objectives
To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source.Results
Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml?1 within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 °C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V 0.Conclusions
The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.16.
Junjie Zhang Yimin Shang Entao Wang Wenfeng Chen Philippe de Lajudie Benyin Li Chen Guo Xu Yang Jianqiang Zheng Chunzeng Liu 《Plant and Soil》2018,425(1-2):201-215
Background and aims
Common bean (Phaseolus vulgaris L.) nodulates with a wide range of rhizobia. Amongst these is Bradyrhizobium, which is inefficient but able to induce profuse nodulation on this crop. Based on this observation, we tested whether co-inoculating bradyrhizobia with a more standard common bean symbiont, Rhizobium tropici, could stimulate growth and nodulation of common bean, thus contributing to a more effective symbiosis.Methods
Rhizobium tropici was co-inoculated with two Bradyrhizobium strains applied at three different doses (104, 106, and 108 CFU seed?1) under sterile conditions, and at a single dose (108 CFU seed?1) in non-sterile soil. Plant biomass, nodulation, and N accumulation in plant tissues were evaluated.Results
Co-inoculated plants produced more nodules, and accumulated more shoot dry biomass and nitrogen than plants inoculated with R. tropici alone under gnotobiotic conditions. Significant responses were observed at the highest inoculum dose and a significant correlation between dose and shoot dry weight was observed. Co-inoculation increased biomass and N accumulation in non-sterile soil, although with a smaller magnitude.Conclusions
Altogether, our findings suggest that the co-inoculation with bradyrhizobia contributed to an improved symbiotic interaction between R. tropici and common beans.17.
Pegah Amiri Azar Shahpiri Mohammad Ali Asadollahi Fariborz Momenbeik Siavash Partow 《Biotechnology letters》2016,38(3):503-508
Objectives
To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.Results
Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l?1.Conclusions
Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.18.
Chunqiu He Wen Dong Jing Li Yanpeng Li Chao Huang Yanling Ma 《Biotechnology letters》2017,39(9):1381-1388
Objectives
To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.Results
Strain DAB had a higher yield of 17.3 g rhamnolipids l?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m?1 with CMC of 90 mg l?1. The predominant rhamnolipid congeners were Rha–C10–C10 and Rha–Rha–C10–C10 detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.Conclusion
Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.19.
Shan Li Lingyan Li Xiangfeng Xiong Xiuling Ji Yunlin Wei Lianbing Lin Qi Zhang 《Biotechnology letters》2018,40(7):1109-1118
Objective
This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation.Methods
Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively.Results
The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5 mM but inhibited by Cu2+ or Zn2+ (p?<?0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703 mM and 156.25 μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15?±?0.24 vs. 2.17?±?0.31 g/L, p?<?0.01).Conclusions
The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.20.