Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae / 中国中药杂志

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Engineering progress in microbial production of polyhydroxyalkanoates / 生物工程学报

Polyhydroxyalkanoates (PHAs) are polymers obtained by esterification of hydroxy fatty acid monomers. Due to similar mechanical characteristics of traditional petroleum-based plastics, 100% biodegradability and biocompatibility, PHAs are considered to be one of the most potential green materials. However, the application and promotion of PHAs as a green and environmentally friendly material are difficult because of the high production costs. This article focuses on the current methods to reduce production cost of PHAs effectively, such as cell morphology regulation, metabolic pathway construction, economic carbon source utilization and open fermentation technology development. Despite most research results are still limited in laboratory, the research methods and directions provide theoretical guidance for the industrial production of economic PHAs.

Construction and high-density fermentation of alkaline pectate lyase high-yield yeast / 生物工程学报

Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.

High density process to cultivate Lactobacillus plantarum biomass using wheat stillage and sugar beet molasses

Background: Owing to the growing interest in biofuels, the concept of a biorefinery where biomass is converted to a variety of useful products is gaining ground. We here present how distillery waste is combined with a by-product from a sugar production, molasses, to form a medium for the growth of Lactobacillus plantarum with yields and biomass densities comparable with conventional industrial media. Such approach enables a cost-effective utilization of the problematic wastewater from ethanol and a by-product from sugar production. It is the first approach that attempts to find low-cost media for the production of Lactobacillus plantarum biomass. Results: This study suggests that sieved wheat stillage enriched by adding 1.77 g/l yeast extract and 10 percent molasses (v/v), with NH4OH used for pH adjustment, may be used as a media for large-scale cultivation of L. plantarum. Such composition of the medium permits a high density of lactic acid bacteria (1.6 x 10(10) cfu/ml) to be achieved. Conclusions: The use of a fermentation medium consisting of distillery wastewater and molasses to obtain value-added products (such as LAB biomass and lactic acid) is a possible step for classical ethanol production to move towards a biorefinery model production in which all by and waste products are utilized to increase produced values and reduce waste production. This enables a cost-effective utilization of the problematic wastewater from ethanol and sugar production.

High Density Fermentation Control of Pichia pastoris / 中国生物工程杂志

One of the important strategies for improving recombinant protein expression in Pichia pastoris is optimizing control during the high density fermentation process. Some fermentation control has been used and got obviously higher expression level:optimization of basal salt medium composition; using two stage pH and two stage temperature control during cell growth and induction stage; appropriately increasing dissolved oxygen; selecting optimal cell density prior to induction (CWPTI) and specific growth rate prior to induction (?PTI),using limit amount of initial glycerolin the basic media and feeding it during the fermentation with exponential fed-batch Model,selecting appropriate methanol feeding strategy by methanol-limited feeding batch (MLFB),oxygen-limited feeding batch (OLFB),methanol-unlimited feeding batch (MNLFB) or temperature-limited feeding batch (TLFB). Some research suggested that the expression level of the recombinant protein increase 9 times after optimizing the fermentation control.

Expression of recombinant humanized anti-HBsAg Fab fragment in genetically engineered Pichia pastoris in high-density fermentation / 解放军医学杂志

Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment

Optimization of Recombinant E. coli High-density Fermentation for Expressing Human-like CollagenⅡ / 微生物学通报

To study and optimize the fermentation parameters for expressing human-like collagenⅡduring E. coli high-density fermentation. The effects of pH, temperature, dissolved oxygen and induction instant on the cell growth and human-like collagenⅡproduction were investigated to optimize the fermentation conditions. The results demonstrated that the following conditions were beneficial for cell growth and foreign gene expression, controlling pH in phase induction at 6.8 and initial pH at 6.5, maintaining fermentation temperature and dissolved oxygen concentration was controlled at 34?C and 20% respectively, and implementing induction at the later logarithmic growth phase. Under the optimized condition, the cell density and human-like collagenⅡyield could reach 88.4 g/L and 14.2 g/L, respectively.

High-density Expression of Recombinant Porcine Insulin Precursor by Pichia pastoris / 微生物学通报

The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e~(0.1907t).Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase Only 80% ammonia and 70% methanol were used by cell in induction phase.By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit- ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen(DO)and.carbon source,respectively.When scaling the result of shake-flask to 501.fermenter,the control strategy was adapted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72 g/L.