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.

Summary: The XylS/ Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW 0 is widely used for regulated low‐ and high‐level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose‐dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β‐lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5′ untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/ Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/ Pm system.

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.

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l^–1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.

The facultative methylotroph Bacillus methanolicus MGA3 has previously been genetically engineered to overproduce the amino acids L-lysine and L-glutamate and their derivatives cadaverine and γ-aminobutyric acid (GABA) from methanol at 50°C. We here explored the potential of utilizing the sugar alcohol mannitol and seaweed extract (SWE) containing mannitol, as alternative feedstocks for production of chemicals by fermentation using B. methanolicus. Extracts of the brown algae Saccharina latissima harvested in the Trondheim Fjord in Norway were prepared and found to contain 12–13 g/l of mannitol, with conductivities corresponding to a salt content of ∼2% NaCl. Initially, 12 B. methanolicus wild type strains were tested for tolerance to various SWE concentrations, and some strains including MGA3 could grow on 50% SWE medium. Non-methylotrophic and methylotrophic growth of B. methanolicus rely on differences in regulation of metabolic pathways, and we compared production titers of GABA and cadaverine under such growth conditions. Shake flask experiments showed that recombinant MGA3 strains could produce similar and higher titers of cadaverine during growth on 50% SWE and mannitol, compared to on methanol. GABA production levels under these conditions were however low compared to growth on methanol. We present the first fed-batch mannitol fermentation of B. methanolicus and production of 6.3 g/l cadaverine. Finally, we constructed a recombinant MGA3 strain synthesizing the C30 terpenoids 4,4′-diaponeurosporene and 4,4′-diapolycopene, experimentally confirming that B. methanolicus has a functional methylerythritol phosphate (MEP) pathway. Together, our results contribute to extending the range of both the feedstocks for growth and products that can be synthesized by B. methanolicus.