Less Is More: Toward a Genome-Reduced Bacillus Cell Factory for “Difficult Proteins”

Abstract

The availability of complete genome sequences and the definition of essential gene sets were fundamental in the start of the genome engineering era. In a recent study, redundant and unnecessary genes were systematically deleted from the Gram-positive bacterium Bacillus subtilis, an industrial production host of high-value secreted proteins. This culminated in strain PG10, which lacks about 36% of the genome, thus representing the most minimal Bacillus chassis currently available. Here, we show that this “miniBacillus” strain has synthetic traits that are favorable for producing “difficult-to-produce proteins”. As exemplified with different staphylococcal antigens, PG10 overcomes several bottlenecks in protein production related to the secretion process and instability of the secreted product. These findings show for the first time that massive genome reduction can substantially improve secretory protein production by a bacterial expression host, and underpin the high potential of genome-engineered strains as future cell factories.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…This strategy enabled the removal of unnecessary proportions of genome at large scale without causing palpable defects in the target organisms (except in a few instances [135,144]) and sometimes yielded improved cellular performances in terms of growth rates, cell density, transformation efficiency, and protein productivity compared with their wild-type counterparts. With an unintended discovery of the desirable properties that synthetic minimal genomes may convey in microbial production, several studies have explored their potential for industrial applications for recombinant protein production [145][146][147][148]. The following sections have been outlined to elaborate in detail on cellular mechanisms underlying improved performance in genome-reduced strains and some of the practical examples of the minimal genome expression system, and also to discuss potential applications of genome-reduced strains in heterologous protein expressions.…”

“…The metabolic burden imposed by expressing heterologous genes arises from competition for shared but limited cellular resources between exogenous genes and host metabolism. Thus, removal of host genes that are nonessential translates to an overall decrease in the resource cost spent on now-absent host metabolism, which then reduces the net metabolic load [159] and allows for efficient resource utilization and protein translation ( Figure 2) [24,137,145]. In addition, reduced genomic complexity in the minimal cells may help to curtail regulatory interference with the targeted or exogenous metabolic pathways [114], reducing the number of compounding variables that pose unknown effects on heterologous expression.…”

“…Empirically, B. subtilis MBG874 strain that underwent a 20% genome reduction exhibited increased productivity of episomally expressed proteins up to 2.5-fold higher than that of the wild-type B. subtilis 168 ( Table 2) [113]. In another instance, mini-Bacillus strains that retained only two-thirds of the B. subtilis 168 genome, with extra re-engineering effort, were able to stably express four individual staphylococcal antigens that are recalcitrant for heterologous protein expression in the existing B. subtilis chassis ( Table 2) [145]. Genome-streamlined L. lactis chassis NZ9000 specializing in the production of many membranous and disulfide-containing proteins also exhibited superior growth rate, biomass yield, and nutrition utilization capacity [139].…”

Membrane Modulation of Super-Secreting “midiBacillus” Expressing the Major Staphylococcus aureus Antigen – A Mass-Spectrometry-Based Absolute Quantification Approach

“…This bacterium is also an attractive host for heterologous protein production due to its excellent fermentation and high product yield capacities (van Dijl and Hecker, 2013). Hence, a minimal strain of B. subtilis was engineered (Reuß et al, 2017) for heterologous production of "difficult proteins" natively secreted by Staphylococcus aureus, Aguilar and colleagues demonstrated that these proteins were successfully produced in the genome-reduced, but not in the wild-type strain (Suárez et al, 2019). Nonetheless, information on how the membrane proteome abundances was altered upon the production of this heterologous protein remained unknown.…”

“…Recently, we have published a study describing a method which simultaneously combines the comprehensiveness of shotgun-MS and the accuracy of targeted-MS, allowing the calculation of absolute membrane protein abundances in a living organism (Antelo-Varela et al, 2019). In the study presented here, we provide absolute membrane protein concentrations of the genome-reduced B. subtilis strain (midiBacillus) IIG-Bs27-47-24 expressing IsaA (Suárez et al, 2019). To engineer this strain, 1401 genes were systematically deleted from the parental strain B. subtilis 168, which represents a genome reduction of 30.95% (Reuß et al, 2017).…”