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Microbial processes can produce a wide range of compounds; however, producing complex and long chain hydrocarbons remains a challenge. Aldol condensation offers a direct route to synthesize these challenging chemistries and can be catalyzed by microbes using aldolases. Deoxyribose-5-phosphate aldolase (DERA) condenses aldehydes and/or ketones to β-hydroxyaldehydes, which can be further converted to value-added chemicals such as a precursor to cholesterol-lowering drugs. Here, we implement a short, aldolase-based pathway in Escherichia coli to produce (R)-1,3-BDO from glucose, an essential component of pharmaceutical products and cosmetics. First, we expressed a three step heterologous pathway from pyruvate to produce 0.3 g/L of (R)-1,3-BDO with a yield of 11.2 mg/g of glucose in wild-type E. coli K12 MG1655. We used a systems metabolic engineering approach to improve (R)-1,3-BDO titer and yield by: 1) identifying and reducing major by-products: ethanol, acetoin, and 2,3-butanediol; 2) increasing pathway flux through DERA to reduce accumulation of toxic acetaldehyde. We then implemented a two-stage fermentation process to improve (R)-1,3-BDO titer by 8-fold to 2.4 g/L and yield by 5-fold to 56 mg/g of glucose (11% of maximum theoretical yield) in strain BD24, by controlling pH to 7 and higher dissolved oxygen level. Furthermore, this study highlights the potential of the aldolase chemistry to synthesize diverse products directly from renewable resources in microbes., (Copyright © 2018. Published by Elsevier Inc.)

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN alpha-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN alpha-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN alpha-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN alpha-2b remains mostly unchanged at a variety of BSA to IFN alpha-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN alpha-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN alpha-2b secondary structure when BSA is in molar excess., ((c) 2009. Published by Elsevier Ltd. All rights reserved.)