Cell Factory Engineering of Undomesticated Bacillus Strains Using a Modified Integrative and Conjugative Element for Efficient Plasmid Delivery

Abstract

A large number of Bacillus strains have been isolated from various environments and many of them have great potential as cell factories. However, they have been rarely developed as cell factories due to their poor transformation efficiency. In this study, we developed a highly efficient plasmid delivery system for undomesticated Bacillus strains using a modified integrative and conjugative element (MICE), which was designed to be activated by an inducer, prevent self-transfer, and deliver desired plasmids to the recipient cells. The MICE system was demonstrated to successfully introduce a gfp-containing plasmid into all 41 undomesticated Bacillus subtilis strains tested and eight other Bacillus species. The MICE was used to deliver a cytosine base editor (CBE)-based multiplex genome-editing tool for the cell factory engineering of the Bacillus species. The introduced CBE enabled one-step inactivation of the major extracellular protease genes of the tested strains. The engineered strains were used as hosts for heterologous expression of nattokinase, which resulted in various enzyme expression levels. The results suggested that the MICE and CBE systems can be powerful tools for genetic engineering of undomesticated Bacillus strains, and greatly contribute to the expansion of the Bacillus cell factory.

One-Pot Production of 3-Hydroxyphloretin from Phlorizin by Bacillus subtilis Whole-Cell System Expressing CYP102A1

“…Plasmid pDC2 was digested with Eco RI and BsiWI, and the large fragment was combined with the P 5DC24 -CYP102A1 mutant cassettes using a cold-fusion cloning kit (System Biosciences Inc., Mountain View, CA, USA) to produce plasmids pDC5DC24-WT, M10, A32, and A42 (Figure ). The resulting plasmids were introduced into B. subtilis 168 by a previously reported method . The integration of the CYP102A1 mutant genes into the chromosome was confirmed by PCR and sequencing.…”

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains

“…The plasmid backbone for the BIP was modified from that of pSGC2 [ 33 ]. To replace E. coli oriT of pSGC2 with Bacillus oriT for MICE conjugation, the plasmid pA3D-gfpTi [ 24 ] was digested with HindIII and SacI, and the small fragment was ligated with HindIII- and SacI-digested pSGC2 to construct pSGC2i. The DSO region and neomycin resistance gene ( neo ) were amplified from pAD123 and pHCas9 [ 12 ], using pSGC2i-DSO-F2/DSO-R3 and 4iN-neo-F/pSGC2i-neo-R, respectively.…”

“…The homologous arms and P spac - cat fragments were cloned into pSGC2iN or pSGC4iN using the Golden Gate assembly. After introducing the resulting plasmid into MICEaRep as a donor strain, it was introduced to target recipients according to a previously reported conjugation method [ 24 ].…”

“…Electroporation or conjugation from an E. coli donor to a Bacillus recipient has been used for the transformation of wild-type Bacillus strains. However, many wild-type strains cannot be transformed using either method [ 24 ]. Therefore, an efficient method for plasmid delivery with a wide host range is required to genetically modify wild-type Bacillus strains.…”

“…Recently, an integrative and conjugative element (ICE)-based DNA transfer method has demonstrated wide host-range characteristics [ 25 ]. In addition, a modified ICE (MICE) capable of delivering plasmids from Bacillus donors to a wide range of undomesticated Bacillus strains has been reported [ 24 ]. However, in the MICE system, both the donor and recipient are Bacillus cells.…”