Riboflavin overproduction from diverse feedstocks with engineered Corynebacterium glutamicum.

Riboflavin overproduction by Corynebacterium glutamicum was achieved by screening synthetic operons, enabling fine-tuned expression of the riboflavin biosynthetic genes ribGCAH. The synthetic operons were designed by means of predicted translational initiation rates of each open reading frame, with the best-performing selection enabling riboflavin overproduction without negatively affecting cell growth. Overexpression of the fructose-1,6-bisphosphatase (fbp) and 5-phosphoribosyl 1-pyrophosphate aminotransferase (purF) encoding genes was then done to redirect the metabolic flux towards the riboflavin precursors. The resulting strain produced 8.3 g/L of riboflavin in glucose-based fed-batch fermentations, which is the highest reported riboflavin titer with C. glutamicum. Further genetic engineering enabled both xylose and mannitol utilization by C. glutamicum, and we demonstrated riboflavin overproduction with the xylose-rich feedstocks rice husk hydrolysate and spent sulfite liquor, and the mannitol-rich feedstock brown seaweed hydrolysate. Remarkably, rice husk hydrolysate provided 30% higher riboflavin yields compared to glucose in the bioreactors. .

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

From Brown Seaweed to a Sustainable Microbial Feedstock for the Production of Riboflavin.

The increasing global demand for food and energy production encourages the development of new production strategies focused on sustainability. Often, microbial bioprocesses rely on food or feed competitive feedstocks; hence, there is a trending need for green substrates. Here, we have proven the potential of brown seaweed biomass as microbial feedstock on account of its content of mannitol and the glucose polymer laminarin. Our host, Corynebacterium glutamicum, was engineered to enable access to mannitol as a carbon source through the heterologous expression of the mannitol-specific phosphotransferase system and the mannitol-1-phosphate-5-dehydrogenase from Bacillus subtilis. Overproduction of riboflavin was coupled with mannitol and glucose consumption via constitutive overexpression of the biosynthetic riboflavin operon ribGCAH from C. glutamicum. Brown seaweed extract and brown seaweed hydrolysate from Laminaria hyperborea, containing mannitol and glucose, were used as a carbon source for flask and bioreactor fermentations. In a seaweed-based fed-batch fermentation, the riboflavin final titer, yield, and volumetric productivity values of 1,291.2 mg L-1, 66.1 mg g-1, and 17.2 mg L-1 h-1, respectively, were achieved.

Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy.

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.

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus.

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.

Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus.

Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5' untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.

Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications.

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.

Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus.

BACKGROUND: 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. 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. RESULTS: Native 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. CONCLUSION: A 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.

Functional characterization of three Azotobacter chroococcum alginate-modifying enzymes related to the Azotobacter vinelandii AlgE mannuronan C-5-epimerase family.

Bacterial alginate initially consists of 1-4-linked ß-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli.

BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS: We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173-2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS: Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

Production of Value-Added Chemicals by Bacillus methanolicus Strains Cultivated on Mannitol and Extracts of Seaweed Saccharina latissima at 50°C.

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.

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

BACKGROUND: The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus. RESULTS: The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts. CONCLUSIONS: While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.

Special Issue: Recombinant Protein Expression in Microorganisms.

Microorganisms are widely used in industrial biotechnology as cell factories for the sustainable production of a wide range of compounds and chemicals [...].

Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production.

BACKGROUND: Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield. RESULTS: Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts. CONCLUSIONS: The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.

Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus.

Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activity was decreased to about 25 % as shown in H2O2 drop assays and by determination of specific catalase activity in crude extracts. Our results support the predicted functions of Spo0A in sporulation and biofilm formation, of MtlD for mannitol catabolism, and of catalase in hydrogen peroxide dismutation. Thus, CRISPR interference as developed here serves as basis for the functional characterization of B. methanolicus physiology as well as for its application in biotechnology.

Corrigendum: Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

[This corrects the article DOI: 10.3389/fmicb.2016.01481.].

Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae.

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 XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.

The XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 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 ß-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.

A novel expression system for lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7-22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42-1298 mg per L of culture; 3.8-11.6 mg per OD600nm unit) as in shaking flasks, and showing the scale-up potential of the system.