Engineering of Saccharomyces cerevisiae for the production of (+)-ambrein

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.     

重组猪源抗菌肽Cecropin P1在毕赤酵母中的分泌表达与活性研究

为构建活性表达重组猪源抗菌肽Cecropin P1的酵母基因工程菌,采用重叠区基因扩增拼接法设计并合成其编码基因,分别载入分泌型表达载体pPICZαB和pGAPZαA中,酶切线性化后电穿孔导入酵母细胞进行整合,经高拷贝整合子筛选以及表型筛选获得高效表达抗菌肽的重组菌株,经培养条件优化确定摇瓶发酵抗菌肽的最佳收获时间,据...     

Influence of pH on recombinant human growth hormone production by Pichia pastoris

BACKGROUND: Effect of pH on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut⁺ was investigated at pH = 4.2, 5.0, 5.5, and 6.0. RESULTS: The highest cell concentration was obtained at pH = 6.0 with 53 g L^?1, while the highest rhGH concentration was attained at pH = 5.0 as 0.27 g L^?1. Total protease secretion increased with increase in pH and with the cultivation time. Oxygen uptake rate increased with increasing pH up to pH = 6.0, having the maximum value, 37 mmol m^?3 s^?1, at pH = 5.5. K_La values were similar at all the conditions, having a maximum value of 0.14 s^?1 at pH = 5.0. Taking the final rhGH concentration into account, the most favourable pH was 5.0; where AOX1 expression level showed a similar trend to AOX activity profiles, having the highest value of 9.4 × 10¹⁰ copy mg^?1 CDW at t = 15 h; in parallel to AOX1 expression profile, hGH expression level increased until t = 15 h, with the highest value of 4.0 × 10¹⁰ copy mg^?1 CDW, where a sharp increase in rhGH concentration was obtained. The expression levels of pep4, prb1 and prc1 genes, responsible from the production of proteinase A, proteinase B and, carboxypeptidase Y, were parallel to each other. CONCLUSION: Since it was shown that pH is a crucial operating parameter in fermentation processes using P. pastoris, keeping pH constant at its determined optimum value, pH = 5.0, during the bioprocess is vital in terms of recombinant protein production. Copyright © 2010 Society of Chemical Industry     

Neutral CeUulase Production by Recombinant Pichia pastoris

通过对6个重组毕氏酵母转化子的产酶试验,筛选出一个优良转化子。在摇瓶条件下对该重组毕氏酵母的发酵过程进行了研究,得到适宜的培养条件为：250 mL三角瓶装液量30 mL,培养基起始pH4.8,此后不加控制,接种量10%,发酵过程中每隔24h补加1%的甲醇对产酶有明显的促进作用,中性纤维素酶活力可达1 144U/mL。研究结果表明：重组毕氏酵母中性纤维素酶的适宜催化条件为：50℃、pH 6.0;Zn2＋和Mg2＋对该酶有激活作用,Fe2＋、Fe3＋和Cu2＋对该酶有抑制作用。牛仔服水洗试验结果显示：重组毕氏酵母中性纤维素酶可以明显降低靛蓝对牛仔服的反染作用,其工业应用前景良好。