Abstract
BackgroundThe suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing. The facultative methylotroph Bacillus methanolicus is gaining interest as a thermophilic cell factory for production of value-added products from methanol. The aim of this study was to expand the capabilities of B. methanolicus as a microbial cell factory by establishing a system for secretion of recombinant proteins.ResultsNative and heterologous signal peptides were tested for secretion of α-amylases and proteases, and we have established the use of the thermostable superfolder green fluorescent protein (sfGFP) as a valuable reporter protein in B. methanolicus. We demonstrated functional production and secretion of recombinant proteases, α-amylases and sfGFP in B. methanolicus MGA3 at 50 °C and showed that the choice of signal peptide for optimal secretion efficiency varies between proteins. In addition, we showed that heterologous production and secretion of α-amylase from Geobacillus stearothermophilus enables B. methanolicus to grow in minimal medium with starch as the sole carbon source. An in silico signal peptide library consisting of 169 predicted peptides from B. methanolicus was generated and will be useful for future studies, but was not experimentally investigated any further here.ConclusionA functional system for recombinant production of secreted proteins at 50 °C has been established in the thermophilic B. methanolicus. In addition, an in silico signal peptide library has been generated, that together with the tools and knowledge presented in this work will be useful for further development of B. methanolicus as a host for recombinant protein production and secretion at 50 °C.
Highlights
The suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing
Selection of candidate α‐amylases and proteases for recombinant production and secretion in Bacillus methanolicus In this study, α-amylases and proteases were chosen as two potential groups of reporter proteins since they are likely to posess native signal sequences, are easy to assay and industrially relevant
Even though the α-amylases chosen for this study were derived from closely related organisms, their amino acid sequences differ substantially from the B. methanolicus-derived Amy, with sequence similarities of 22.47%, 27.62% and 24.86% for AmyS, AmyE and amylases from B. licheniformis (AmyL), respectively, ensuring that different signal peptides and different model proteins were tested in B. methanolicus
Summary
The suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing. Heat resistant enzymes have innate advantages over their mesophilic counterparts, including increased temperature stability and resistance to proteolytic cleavage by proteases [5]. Thermophilic species such as Bacillus licheniformis and Geobacillus stearothermophilus are utilized as donors of industrially important enzymes like α-amylases and proteases, which are extensively used for detergent production [6, 7]. Many of these enzymes can fold properly at temperatures 60 °C below their physiological conditions, and have the same. Many thermophilic organisms grow slowly and have low biomass productivities, making them poor choices as hosts for industrial production of proteins, B. methanolicus can reach specific growth rates of 0.46 h−1, making it a good choice as a thermophilic host for protein production [13, 14]
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