High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine / 生物工程学报

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.

Construction of a highly efficient synthetic lycopene engineered Saccharomyces cerevisiae / 生物工程学报

Lycopene, as a high value-added terpene compound, has been widely concerned by researchers at home and abroad. Firstly, the ability of lycopene synthesis of Saccharomyces cerevisiae model strains S288c and YPH499 was analyzed and compared. The results showed that YPH499 was more suitable for lycopene synthesis as yeast chassis. Subsequently, the effects of constitutive promoters GPDpr, TEF1pr and inducible promoters GAL1pr, GAL10pr on Lycopene synthesis were compared. The results showed that when GPDpr and TEF1pr were used as promoters of crtE, crtB and crtI in lycopene synthesis pathway, the production of lycopene was 15.31 mg/L after 60 h fermentation in shaking flask. When GAL1pr and GAL10pr were used as promoters, the production was 123.89 mg/L, which was 8.09 times higher. In addition, the methylvaleric acid (MVA) pathway was further modified to overexpress the key enzyme gene of N-terminal truncation, tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase). The lycopene production was 265.68 mg/L, and the yield per cell was 72.79 mg/g. The Saccharomyces cerevisiae strain designed and constructed in this study can express lycopene in high yield per cell, thus could be used in the industrial production of lycopene after further construction and optimization.

Development and verification of an FLP/FRT system for gene editing in Bacillus licheniformis / 生物工程学报

Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.

Relationship between mycelium morphology and laccase production of Pleurotus ferulae in submerged cultivation / 生物工程学报

In this study, the relationship between mycelium morphology and laccase production was studied. The results indicated that the morphology of P. ferulae pellets was changed when glass beads were added. Laccase production showed higher with spherical mycelium than with filamentous or flocculent mycelium. In addition, the spherical mycelium with a diameter of 0.2-0.4 mm highly affected laccase production. Effect of the composition of culture medium on pellets was investigated and results indicated that various concentrations of glucose, corn meal and wheat bran were important to the formation of pellets in diameter of 0.2-0.4 mm. Besides nutrients, the addition of non-nutritional substrates influenced the distribution of P. ferulae pellets. However, the production of laccase was not promoted by non-nutritional substrates.

Cloning, expression of phospholipase A1 from Serratia liquefaciens and auto-induction fermentation by lactose / 生物工程学报

To produce recombinant phospholipase A(1) (PLA(1)) by Escherichian coli, the pla gene encoding PLA(1) was amplified from Serratia liquefaciens by PCR and cloned into two vectors pET20-b(+) and pET28-a(+). The two recombinant plasmids were then transformed into E. coli BL21 (DE3) individually to express PLA(1). E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA(1) with an activity of 40.8 U/mL in batch cultivations of shaken flasks by auto-induction, which was accounted for 91% of total enzyme activity. On the basis of primal auto-induction medium, the optimized fermentation medium of PLA(1) contained tryptone 10 g/L, yeast extract 5 g/L, glucose 0.8 g/L, lactose 5 g/L, Na2HPO4 25 mmol/L, KH2PO4 25 mmol/L and 1 mmol/L MgSO4 (final concentration). Glycine (7.5 g/L) was added 6 h after inoculated. After incubated at 37 degrees C for 24 h, extracellular enzyme activity reached 128.7 U/mL.

Metabolic engineering of wild acid-resistant yeast for L-lactic acid production / 生物工程学报

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.

Asymmetric biosynthesis of d-pseudoephedrine by recombinant Bacillus subtilis / 生物工程学报

In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl- 1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.

Metabolic engineering for improving ethanol fermentation of xylose by wild yeast / 生物工程学报

One yeast strain, which was isolated from 256 natural samples, was found to be able to utilize D-xylose effectively. On the basis of assimilation physiological and molecular biological tests, the yeast strain was identified as a strain of Candida tropicalis. Furthermore, metabolic engineering breeding strategy was applied to change the metabolic flux in order to increase ethanol productivity. In this study, the C. tropicalis was used as the host strain and the plasmid pYX212-XYL2, which was formerly constructed for over expression of XYL2 gene encoding xylitol dehydrogenase (XDH) from Pichia stipitis, was used as the backbone of the recombinant vector. A hygro gene was inserted into downstream position of XYL2 gene, meanwhile, the result plasmid pXY212-XYL2-Hygro transformed into C. tropicalis by electroporation. Thus, a recombinant yeast C. tropicalis XYL2-7 was obtained through hygromycin B resistance screening and its specific XDH activity was 0.5 u/mg protein, which was 3 times more than that of the parent strain. Additionally, the recombinant yeast was applied in the fermentation of xylose. Compared with the parent yeast, it was concluded that the xylitol yield in the broth decreased by 3 times, however, the ethanol yield increased by 5 times. The feasibility of ethanol production from xylose by C. tropicalis was firstly studied in this paper. These research results are helpful to advance the bioconversion of renewable resources (e. g. straw, wheat bran, and husk) to fuel ethanol.

Experience on Laparoscopic Total Extraperitoneal Repair of Inguinal Hernia / 中国微创外科杂志

Objective To discuss the value of laparoscopic total extraperitoneal repair(TEP) for inguinal hernia.MethodsBetween 2005 and 2007,82 consecutive patients with inguinal hernia at 97 sides underwent TEP by laparoscopy in our hospital.Among the cases [76 men and 6 women with a mean age of 52 years(21 to 88)],9 had unilateral direct inguinal hernia,50 showed unilateral indirect inguinal hernia,and 9 suffered from bilateral indirect inguinal hernia,6 cases were found having bilateral indirect inguinal hernia complicated with direct hernia,and 8 were recurrent cases of indirect inguinal hernia.Results In the patients,5 showed massive postoperative abdominal adhesion;one of them was converted to open surgery because of severe injury to the peritoneum,the other 4 were treated by continuous suture using 5-0 absorbable thread.The operation time for laparoscopic surgery ranged from 30 to 180 minutes(58 minutes for unilateral operation,and 97 minutes for bilateral operation on average).No patient needed analgesics after the procedure.The mean hospital stay for this series was 7 days(range: 4 to 12).Nine patients developed seromas of the scrotum,and was then cured by local drainage and physical therapy.The 82 patients were followed up for 13 to 38 months with a mean of 26 months,during which no one had recurrence.Conclusion As a safe and minimally invasive procedure,which leads to less trauma and pain,as well as quick recovery,TEP is especially suitable for patients with recurrent or bilateral hernias.