Formaldehyde metabolism is prevalent in all organisms, where the accumulation of formaldehyde can be prevented through the activity of dissimilation pathways. Furthermore, formaldehyde assimilatory pathways play a fundamental role in many methylotrophs, which are microorganisms able to build biomass and obtain energy from single- and multicarbon compounds with no carbon–carbon bonds. Here, we describe how formaldehyde is formed in the environment, the mechanisms of its toxicity to the cells, and the cell’s strategies to circumvent it. While their importance is unquestionable for cell survival in formaldehyde rich environments, we present examples of how the modification of native formaldehyde dissimilation pathways in nonmethylotrophic bacteria can be applied to redirect carbon flux toward heterologous, synthetic formaldehyde assimilation pathways introduced into their metabolism. Attempts to engineer methylotrophy into nonmethylotrophic hosts have gained interest in the past decade, with only limited successes leading to the creation of autonomous synthetic methylotrophy. Here, we discuss how native formaldehyde assimilation pathways can additionally be employed as a premise to achieving synthetic methylotrophy. Lastly, we discuss how emerging knowledge on regulation of formaldehyde metabolism can contribute to creating synthetic regulatory circuits applied in metabolic engineering strategies.

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 g/L to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Worldwide, the use of methane is limited to generating power, electricity, heating, and for production of chemicals. We believe this valuable gas can be employed more widely. Here we review the possibility of using methane as a feedstock for biotechnological processes based on the application of synthetic methanotrophs. Methane monooxygenase (MMO) enables aerobic methanotrophs to utilize methane as a sole carbon and energy source, in contrast to industrial microorganisms that grow on carbon sources, such as sugar cane, which directly compete with the food market. However, naturally occurring methanotrophs have proven to be difficult to manipulate genetically and their current industrial use is limited to generating animal feed biomass. Shifting the focus from genetic engineering of methanotrophs, towards introducing metabolic pathways for methane utilization in familiar industrial microorganisms, may lead to construction of efficient and economically feasible microbial cell factories. The applications of a technology for MMO production are not limited to methane-based industrial synthesis of fuels and value-added products, but are also of interest in bioremediation where mitigating anthropogenic pollution is an increasingly relevant issue. Published research on successful functional expression of MMO does not exist, but several attempts provide promising future perspectives and a few recent patents indicate that there is an ongoing research in this field. Combining the knowledge on genetics and metabolism of methanotrophy with tools for functional heterologous expression of MMO-encoding genes in non-methanotrophic bacterial species, is a key step for construction of synthetic methanotrophs that holds a great biotechnological potential.

Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5′-ends. Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5′-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5′-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts. The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B. methanolicus, B. coagulans, B. smithii, B. licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.