Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

Abstract

Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.Due to recent advances in genetic engineering technology, a variety of useful substances, including enzymes, have been produced industrially by use of microorganisms. To further improve production efficiencies on an industrial scale, several approaches, such as mutational breeding, have been used to generate hyperproducing microbial cells. In addition, strategies for genome reduction (18), which represents a relatively new field in synthetic genomics, have been used with Escherichia coli (25,44) and Bacillus subtilis (3,39,54) to investigate microbial genomic architecture and to improve characteristics relevant to protein production.B. subtilis, a Gram-positive sporiferous bacillus, is an attractive organism for industrial use for a variety of reasons, including its high growth rate, protein secretion ability, and GRAS (generally regarded as safe) status (46, 49). B. subtilis is also one of the best-characterized model microorganisms, by biochemical, genetic, and molecular biological studies. Furthermore, the complete genomic sequence of B. subtilis strain 168 has been determined, facilitating genetic engineering of this industrially useful strain (4, 35).In the 4.2-Mb genome of B. subtilis strain 168, only 271 of the 4,106 identified genes are indispensable for the growth of this organism in rich medium (33). In addition, B. subtilis has numerous genes that are activated only under specific conditions or in response to environmental stresses. Therefore, under controlled cond...

Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

“…This is not unusual, as described for B. subtilis, where not the genome reduction by 20% in general but mainly the deletion of the rocDEF-rocR region was shown to be responsible for an elevated protein production. This region is involved in arginine degradation, and the deletion causes drastic changes in glutamate metabolism, leading to improved cell yields (45). In the case of C. glutamicum, the relevant operon codes for an RM system first described in the late 1980s (46,47).…”

High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

“…Deletion of this genomic region significantly increased the production of cellulase Egl-237 and M-protease [7,8], with subsequent analyses revealing that deletion of the rocDEF-rocR region was responsible for the enhanced enzyme production [11]. However, here, we found that deletion of the rocDEF-rocR region caused a significant decrease in the production level of AmyK38 (Additional file 1: Figure S1).…”

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3

“…Enlightened by that, we attempted to enhance intracellular glutamate synthesis by genetically modifying related genes. Deletion of RocR (a regulator participating in glutamate degradation by activating the gene encoding glutamate dehydrogenase, rocG) was found to increase the flux from 2-oxoglutarate to glutamate, and then lead to increased synthesis of the other amino acids via transamination in B. subtilis 168 [13]. Besides rocR, rocG, gudB and odhA were also targeted in this study.…”