An integrated biotechnology platform for developing sustainable chemical processes.

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.

Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil.

BD16449 lipase is the product of a phospholipid-specific lipase gene expressed in the yeast Pichia pastoris strain DVSA-PLC-004. This type C phospholipid lipase (EC 3.1.4.3) is intended for use in the degumming of edible vegetable oil. BD16449 lipase was tested as a refined test article preparation (DV16449) for its effects on genotoxicity and in acute, inhalation, and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to 2000 mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000 mg/kg/day) resulted in a safety margin of 133,000 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.015 mg/kg/day. When adjusted for total organic solids (TOS), the highest oral dose tested in vivo (NOAEL of 1680 mg TOS/kg/day) resulted in a safety margin of 18,300 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.092 mg TOS/kg/day [corrected] There was no toxicity reported for any of these studies including additional safety studies. A review of the literature indicates that P. pastoris fulfills recognized safety criteria pertinent to microbial production strains used in the manufacture of food enzyme preparations. The results of the toxicity studies presented herein attest to the safety of BD16449 lipase for use in the degumming of edible vegetable oil.

Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement.

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62 degrees C for 1 h and 27% of its initial activity after 10 min at 85 degrees C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

Safety evaluation of a phytase, expressed in Schizosaccharomyces pombe, intended for use in animal feed.

BD006 phytase is the product of the Escherichia coli B app A gene expressed in Schizosaccharomyces pombe strain ASP595-1. This enzyme preparation is intended for use in animal feed applications where it improves the bioavailability of phosphate for monogastric animals. BD006 phytase was tested as an unrefined (DV006U) and a refined (DV006R) preparation for its effects on genotoxicity and in acute, inhalation and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to a high of 2000X for oral in vivo toxicity studies. The highest oral dose tested (2000X) is 2000 times the highest expected consumption of product by poultry or swine (X=50 units of phytase per kg bwt/day). There was no genotoxicity or any in vivo toxicity reported which could be directly related to systemic effects of the product. Other additional safety studies conducted were devoid of any relevant toxicity. The historic safety profile of the production organism S. pombe, and the results of the toxicity studies presented herein, attest to the safety of BD006 phytase for use in animal feed applications.