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Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyse the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anti-cancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimisation of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimisation enhanced total oxygenated taxane titre to 39.0±5.7 mg/L and total taxane product titres were comparable at micro and mini-bioreactor scale at 95.4±18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titre to 19.2 mg/L, comparable to the 23.8±3.7 mg/L achieved at microscale. A combination of bioprocess optimisation and increased GC-MS resolution at 1L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titre of 3.7 mg/L. Total oxygenated taxane titres were improved 2.7-fold at this scale to 78 mg/L, the highest reported titre in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems. This article is protected by copyright. All rights reserved.

Groundwater is becoming an increasingly important drinking water source. However, the use of groundwater for potable purposes can lead to chronic human exposure to geogenic contaminants, for example, uranium. Nanofiltration (NF) and reverse osmosis (RO) processes are used for drinking water purification, and it is important to understand how contaminants interact with membranes since accumulation of contaminants to the membrane surface can lead to fouling, performance decline and possible breakthrough of contaminants. During the current study laboratory experiments were conducted using NF (TFC-SR2) and RO (BW30) membranes to establish the behavior of uranium across pH (3-10) and pressure (5-15 bar) ranges. The results showed that important determinants of uranium membrane sorption interactions were (1) the uranium speciation (uranium species valence and size in relation to membrane surface charge and pore size) and (ii) concentration polarization, depending on the pH values. The results show that it is important to monitor sorption of uranium to membranes, which is controlled by pH and concentration polarization, and, if necessary, adjust those parameters controlling uranium sorption., QC 20160715