Biosynthesis recombinant proteins

Flavanones are converted stereospecifically to the respective (2i ,3i )-dihydroflavonols (DHFs) by flavanone 3(3-hydroxylase. Stereospecificity for (2S)-flavanones has been confirmed by analysis of the recombinant protein. Flavanone 3(3-hydroxylase is commonly abbreviated to F3H, which is what has been used in this chapter, but FHT is also used in the literature, which agrees with the nomenclature for phenylpropanoid biosynthesis proposed in Heller and Forkmann. ... 

UDP-GlcNAc represents the branching point for the bacterial biosynthesis of murein and lipopolysaccharides where secondary nucleotide sugars are involved [329, 358], Starting from fructose-6-P, all enzymes are available as recombinant proteins and enzymatic synthesis of important intermediates seems to be possible. 

The metabolic flux distributions around the intermediate pyruvate for different strains and environmental conditions are summarised in Fig. 12. This part of the metabolism has been shown to be an important node for the interconversion between glycolytic C3 metabolites and C4 metabolites of the tricarboxylic acid (TCA) cycle. The different anaplerotic reactions are of special importance for the production of recombinant proteins as they provide precursors, such as oxaloace-tate, for amino acid biosynthesis. Due to that, the flux distribution is noticeably affected by both the cultivation conditions and the carbon source used which indicates flexible adaptation to the environmental situation. The flux from pyruvate to oxaloacetate through the reaction catalysed by pyruvate carboxylase was found to be the main anaplerotic pathway in B. megaterium. 

As noted earlier, experiments leading to a bacterially expressed recombinant protein from chromosome 4 of Arabidopsis with the properties of MIOase have provided opportunity to test the lull potential for AsA biosynthesis through the inversion pathway (Lorence et al., 2004). It remains to be determined whether this route is ancillary to the D-mannose/L-galactose (alternatively, L-gulose) pathway or is a major source of AsA via MIOase in plants. 

Baumann, K., Adelantado, N., Lang, C., Mattanovich, D., and Ferrer, P. (2011) Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris. Microb. Cell Fact, 10, 93. 

The biochemistry and enzymology of CHS and its closely related plant PKSs have been extensively studied [72, 75]. The native enzymes have been isolated from various plants [70,71,73,74,124,125,127,128], and the recombinant proteins have been produced in E. coli and purified [71,76,124,125,132-135,139]. The active site for the condensation reaction has been mapped to a single Cys residue [132]. The dimeric nature of CHS and STS has been established with each homodimer to constitute two active sites [76,127,133]. The subtle differences among the CHS and STS proteins have been compared, which seems to have a profoimd effect on substrate specificity and regiospedfidty for the condensation and cyclization reactions, respectively [74, 127, 128, 133, 134]. The interactions between CHS and other proteins, such as KR [133,136-139] and methylmalonyl CoA-specific condensing enzyme [76], have been examined to account for the biosynthesis of deoxychalcones and methylchalcones. 

Among the important process-related cost drivers are (i) material consumption and (ii) duration of the process. As an example, the cost share of the carbon source (e.g., glucose) for the fermentation (biosynthesis) of a bulk commodity product in a low labor cost environment typically exceeds 70% of the USP cost, while the remaining 30% is distributed over personnel, maintenance, depreciation, and others. This contrasts with high-value specialties such as recombinant proteins, where the cost of the carbon source in fermentation is negligible. In the breakdown shown in the following, material comprises not only the carbon source but also the costs of other consumables and disposables. 

To ascertain whether the recombinant proteins were active, soluble extracts from cells containing the constructs were fractionated by ion exchange chromatography. The fractions were analysed in a complementation assay involving a crude extract of E. coli that had been treated with 200 pM cerulenin to inhibit KASes I, II and IV. That is, fractions were added back to the treated extract, and monitored for restoration of fatty acid biosynthesis by measuring the incorporation of radioactively labelled malonyl-CoA into fatty acids. 

Optimizing Sialylation of Recombinant Proteins by Metabolic Engineering Sialic Acid Biosynthesis... 

Figure 1. Metabolic pathways for sialylation of recombinant proteins expressed in animal cells. Although the precise pathway in CHO or BHK cells has not yet been clearly defined, shown are potential routes for sialic acid biosynthesis based on metabolic studies of other tissues and somatic cell types. Enzymes shown in bold type may be gene targets for overexpression in animal cells to improve sialylation under high productivity conditions.

Optimizing Gaiactosylation of Recombinant Proteins by Metabolically Engineering Galactose Biosynthesis... 

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