1 INTRODUCTION
Directed evolution has proved to be a powerful tool to improve the
activity, stability and substrate specificity of enzymes (Goldsmith et
al. 2017; Romero et al. 2009; Roodveldt et al. 2005; Wong et al. 2004;
Wong et al. 2007; Zeymer et al. 2018). This tool involves two crucial
steps: generation of a library containing sufficient gene variants and
high-throughput screening of library members with desired properties
(Packer et al. 2015; Wong et al. 2006). Because of its high
transformation efficiencies, rapid growth rates, well-established
manipulation approaches (Crameri et al. 1996), Escherichia coliis generally employed as host organism for library creation. However,
proteins are usually expressed in cytoplasm in E. coli , making
the screening of library more difficult if the substrate of the protein
cannot be transported into cell. Therefore, the organisms such asBacillus subtilis and Pichia pastoris that can secret
proteins into medium have been developed as alternative hosts for
library generation (Jiang et al. 2019; Liu et al. 2017; Reetz et al.
2007; Wang et al. 2012).
B. subtilis is an important industrial host for production of
various recombinant proteins due to its GRAS (generally recognized as
safe) status, excellent protein secretion ability and mature
fermentation processes (Commichau et al. 2014; Marcus et al. 2004;
Schumann et al. 2007; Terpe et al. 2006; Yang et al. 2011). AlthoughB. subtilis has well-developed genetic manipulation tools, its
transformation frequency is still much lower than that of E.
coli . Thus, the library of random mutants generated through digestion
and ligation is too small to fulfill the need of direct evolution inB. subtilis . Several strategies have been adopted to address this
limitation. A routine strategy is first constructing the library of
variants in E. coli and then transferring the library intoB. subtilis , which is time-consuming and of low efficiency
(Caspers et al. 2010; You et al. 1996; Zhao et al. 1999). Melnikov et
al. (1999) attempted to clone error-prone PCR (epPCR) product via
marker-replacement recombination with a structurally similar helper
plasmid resident in the transformation recipient. But they found that it
was difficult to recover >103transformants/μg of epPCR product. Given the phenomenon that multimeric
plasmid has much higher (approximately three orders of magnitude)
transformation frequency than that of monomeric plasmid in B.
subtilis (Canosi et al. 1978), Shafikhani et al. (1997) generated large
libraries of random mutants (3×106) in B.
subtilis by PCR-based plasmid multimerization method. In this method,
epPCR product was fused with linearized vector through PCR extension to
generate linear plasmid multimer which could be converted to circular
form through homologous recombination after entering cell; however, each
cell may take several variants, decreasing screening efficiency.
Increasing the number of competent cells is an effective method to
create larger library. To this end, Zhang and Zhang et al. (2011)
constructed a B. subtilis strain SCK6 whose competence can be
induced by artificially controlling the expression of master regulator
ComK. The procedure for preparing supercompetent cell of strain SCK6 was
further improved by Li et al. (2017). The transformation frequencies of
multimeric plasmid, monomeric plasmid and integration plasmid into
strain SCK6 could reach 107, 104 and
105 transformants/μg DNA.
This study aims to construct large mutant library through inserting
epPCR product into the chromosome of B. subtilis , which will
solve the problems of plasmid instability and heterozygosity faced by
multi-copy plasmid mediated strategies (Melnikov et al. 1999; You et al.
2012; Zhang and Zhang 2011). We accomplished the library construction
within one day and the generated library had sufficient mutants
(>105) for the need of direct evolution.