Marco Malten

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B12) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome‐based overexpression of genes involved in vitamin B12 biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysGAcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B12 riboswitch upstream of the cbiXJCDETLFGAcysGAcbiYbtuR operon and the recombinant production of three different vitamin B12 binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B12‐dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B12 concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.

Calculating sugar yields in high solids hydrolysis of biomass.

Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in yield calculations can lead to significant errors. In this paper, a mathematical method was developed for the estimation of liquid volume change and thereafter the sugar yield. The information needed in the calculations include the compositions of the substrate, initial solids loading, initial liquid density, and sugar concentrations before and after hydrolysis. All of these variables are measurable with conventional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in the literature, this method is relatively easy to use and provides good accuracy.

Biomass conversion determined via fluorescent cellulose decay assay.

An example of a rapid microtiter plate assay (fluorescence cellulose decay, FCD) that determines the conversion of cellulose in a washed biomass substrate is reported. The conversion, as verified by HPLC, is shown to correlate to the monitored FCD in the assay. The FCD assay activity correlates to the performance of multicomponent enzyme mixtures and is thus useful for the biomass industry. The development of an optimized setup of the 96-well microtiter plate is described, and is used to test a model that shortens the assay incubation time from 72 to 24h. A step-by-step procedure of the final assay is described.