Abstract
BackgroundSome strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable.ResultsA new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA) production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained.ConclusionThis production level of BLA by B. licheniformis CBBD302(pHY-amyL) is amongst the highest levels in Gram-positive bacteria reported so far.
Highlights
Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable Damylase and alkaline protease production for over 40 years
In order to select a suitable host cell for Bacillus licheniformis D-amylase (BLA) overproduction, following genetic and physiological criteria were applied: 1) the ability of the strain to sporulate should be poor in order to extend the duration of BLA production; 2) the strain should form little or no lichenysin in order to reduce consumption of ATP and the amino acid pool; 3) the strain should not clump during cultivation to maintain efficiency in a production bioreactor; 4) the strain should grow well on either low-cost fermentation medium containing a high substrate concentration appropriate for an industrial process; 5) the strain should contain no native plasmids but be sensitive to kanamycin or tetracyclin to facilitate further genetic manipulation and 6) the strain should be amenable to transformation to enable genetic modification
A strain CBBD302 was developed by deletion of a type I restriction modification systems (RMS) locus in strain CBB0302 by using homolog-mediated recombination according to the method described by Waschkau et al [19]
Summary
Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable Damylase and alkaline protease production for over 40 years. To obtain a high yield in bacterial extracellular enzyme production, the following genetic and physiological properties of the strain are important: a) the metabolic flux for amino acids synthesis and ATP regeneration, b) the cell growth rate and cell density in an inexpensive medium, c) mainly vegetative growth by spore-forming strains, d) secretion capacity for extracellular enzymes, e) long-term preservation in an active form in broth, and f) a high expression level of the specific gene encoding a bacterial extracellular enzyme [3]. Genetic improvement of bacterial extracellular enzyme production is achieved by applying a range of strategies based on molecular cloning tools. These include: 1) enhancement of expression level through amplification of gene copy number [4], codon usage optimization [5], or strong promoters being used to boost gene transcription (page number not for citation purposes)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
