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Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.

Alkene monooxygenase from Xanthobacter autotrophicus Py2 (XAMO) catalyses the asymmetric epoxidation of a broad range of alkenes. As well as the electron transfer components (a NADH-oxidoreductase and a Rieske-type ferredoxin) and the terminal oxygenase containing the binuclear non-haem iron active site, it requires a small catalytic coupling/effector protein, AamD. The effect of changing AamD stoichiometry and substitution with effector protein homologues on the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation has been studied. At sub-optimal stoichiometries, there was a marked change in regioselectivity, but no significant change in epoxidation stereoselectivity. Recombinant coupling proteins from a number of phylogenetically related oxygenases were investigated for their ability to functionally replace AamD. Substitution of AamD with IsoD, the coupling protein from the closely related isoprene monooxygenase, changed the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation, although this was accompanied by a high level of uncoupling. This indicates the importance of coupling protein interaction in controlling the catalytic specificity. Sequence analysis suggests that interaction between Asn34 and Arg57 is important for complementation specificity of the coupling proteins, providing a candidate for site-directed mutagenesis studies.

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.

Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.