Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae / 生物工程学报

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.

Effects of germanium on cell growth, polysaccharide production and cellular redox status in suspension cultures of protocorm-like bodies of Dendrobium huoshanense / 生物工程学报

To solve the problem of low growth rate and metabolism level in suspension cultures of protocorm-like bodies (PLBs) of Dendrobium huoshanense. The effects of germanium on PLB proliferation and accumulation of polysaccharides together with nutrient utilization were investigated and the contents of reducing sugars, soluble proteins, the activities of antioxidant enzymes and redox status of the cells of PLB were analyzed. The results indicated that the optimum concentration of germanium dioxide (4.0 mg/L) significantly enhanced the cell growth and accumulation of polysaccharides, greatly improved contents of reducing sugars and soluble proteins, increased the activities of superoxide dismutase (SOD) and catalase (CAT) but decreased the activity of peroxidase(POD). The cell dry weight and production of polysaccharides were 32.6 g/L and 3.78 g/L, respectively. The analysis of cellular redox status showed that the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in cells and the activity of glutathione reductase were significantly increased by the addition of germanium dioxide. The suitable concentration of germanium dioxide was beneficial to the cell growth and the accumulation of polysaccharides.

Repeated intermittent L-lactic acid fermentation technology by self-immobilized Rhizopus oryzae / 生物工程学报

In order to carry out the process of the repeated intermittent L-lactic acid fermentation by self-immobilized Rhizopus oryzae, we investigated the effect of medium compositions on the morphology of Rhizopus oryzae. In submerged culture, fungi can be grown as broths of freely suspended mycelia and pellets or clumps, the pellets can be immobilized by themselves. The optimum medium composition for the first patch of fermentation was: 120 g/L glucose, 3 g/L NH4NO3, 0.14 g/L KH2PO4, 0.16 g/L NaH2PO4 (the concentration ratio of K+ and Na+ was 1:1). After 72 h fermentation, the physical form of Rhizopus oryzae was mostly uniform pellet with the diameter of 1.0-2.0 mm, the concentration of L-lactic acid was 100.8 g/L, and the conversion rate of glucose was 84%. During 16 batches of repeated fermentation, the L-lactic acid level was above 60.0 g/L and the conversion rate of glucose was 75%. The fermentation time of every batch was 24 h.

Breeding of Actinobacillus succiniogenes mutants with improved succinate production based on metabolic flux analysis / 生物工程学报

It is very important to obtain high yield mutant strains on the base of metabolic flux analysis of Actinobacillus succinogenes S.JST for the industrial bioconversion of succinic acid. The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the flux of succinic acid was 1.78(mmol/g/h) and that the flux of acetic acid and ethanol were 0.60 (mmol/g/h) and 1.04( mmol/g/h), respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 (mmol/g/h), decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 (mmol/g/h), decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 (g/L). The interdiction of Adh and Pta decreased the metabolism of byproducts and the H electron donor was well balanced, thus the succinic acid flux was strengthened by the redundant carbon flux from these byproducts. The mutant strain S.JST2 obtained in this paper deserves being extended to application of industrial fermentation.