No abstract

Serratia marcescens has been proved to be a potential strain for industrial 2,3-butanediol production for its high yield, productivity, and other advantages. In this study, the genes slaA, slaB, slaC, and slaR were successfully cloned which were further confirmed to be encoding acetolactate decarboxylase, acetolactate synthase, 2,3-butanediol dehydrogenase, and a LysR-like regulator, respectively. Unlike in Klebsiella sp. or Klebsiella pneumonie and Vibrio sp. or Vibrio cholerae, the gene slaC is separated from other genes. Then it showed that two regulators, SwrR and SlaR, are in charge of this process by exerting effect on the transcription of genes slaA and slaB. By contrast, the expression of gene slaC is unaffected by the two regulators. It means that these two regulators affect the production of 2,3-butanediol by regulating the production of acetoin. Based on these findings, we successfully accelerated the 2,3-butanediol production by inactivation of gene swrR. The obtained results and further investigations should lead to a more suitable fermentation strategy and strain improvement which would be applicable to the industrial production of 2,3-butanediol.

(3R)-Acetoin and (2R,3R)-2,3-butanediol are important pharmaceutical intermediates. However, until now, the quantity of natural microorganisms with the ability to produce single configuration of optically pure (3R)-acetoin and (2R,3R)-2,3-butanediol is rare. In this study, a meso-2,3-butanediol dehydrogenase encoded by the slaC gene from Serratia marcescens MG1 was identified for meso-2,3-butanediol and (2S,3S)-2,3-butanediol biosynthesis. Inactivation of the slaC gene could significantly decrease meso-2,3-butanediol and (2S,3S)-2,3-butanediol and result in a large quantity of (3R)-acetoin accumulation. Furthermore, a (2R,3R)-2,3-butanediol dehydrogenase encoded by the bdhA gene from Bacillus subtilis 168 was introduced into the slaC mutant strain of Serratia marcescens MG1. Excess (2R,3R)-2,3-butanediol dehydrogenase could accelerate the reaction from (3R)-acetoin to (2R,3R)-2,3-butanediol and lead to (2R,3R)-2,3-butanediol accumulation. In fed-batch fermentation, the excess (2R,3R)-2,3-butanediol dehydrogenase expression strain could produce 89.81 g/l (2R,3R)-2,3-butanediol with a productivity of 1.91 g/l/h at 48 h. These results provided potential applications for (3R)-acetoin and (2R,3R)-2,3-butanediol production.

No abstract

No abstract

The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A-base overhang. The other vector pYEVB, with the same features for blunt-end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X-gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full-length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by Sal I and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time-consuming conventional cloning procedures.

No abstract

BackgroundThe methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs.ResultsA visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology.ConclusionsA novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.