Host For Heterologous Protein Production Research Articles

Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology

In the last years, many research efforts have been applied for the development of filamentous fungi as hosts for heterologous protein production. Aspergillus vadensis CBS 113365, a close relative of the industrial workhorse Aspergillus niger, has been suggested as a more suitable cell factory as it does not acidify the culture medium and produces very low levels of secreted proteases. Therefore, efficient methods and tools that allow the genetic manipulation and exploitation of this biotechnologically relevant fungus are needed. To date, only protoplast-mediated transformation and classical cloning strategies have been implemented for A. vadensis genetic modification, which decreases the exploitation capacity of this fungus at the industrial level. In this study, we have adapted and implemented an Agrobacterium tumefaciens-mediated transformation protocol for A. vadensis for the first time, and applied the FungalBraid system to genetically modify this species by means of synthetic biology. As proof of concept, we have successfully complemented and fluorescently labelled a uridine auxotrophic A. vadensis pyrA- strain and generated A. vadensis mutants carrying the Penicillium expansum-based expression cassette for the heterologous production of the antifungal protein PeAfpA from P. expansum. Even though we have yet to find the conditions that trigger PeAfpA production in this species, the implementation of the ATMT method reported here, along with the application of the FungalBraid system, will greatly aid in this task and will facilitate the exploitation of A. vadensis as a fungal workhorse for protein production for multiple biotechnological applications.

AOX1 promoter-driven expression of yeast-enhanced green fluorescent protein in Pichia pastoris

Green fluorescent protein (GFP) is a widely employed reporter for studying various cellular functions in genetic engineering. Methylotrophic yeast; Pichia pastoris is an excellent host for heterologous protein production. Codon optimized yeast-enhanced green fluorescent protein (yEGFP) has been reported to have higher expression as a yeast reporter which can be used to study many aspects of P. pastoris. The present investigation was focused on studying the yEGFP intracellular expression pattern in the P. pastoris GS115 strain. yEGFP is cloned into the pPICZ A vector under the control of an AOX1 (Alcohol Oxidase) promoter that was induced by adding methanol to the growth medium. However, methanol concentration and time of induction could affect the optimum expression of yEGFP. Hence, those conditions were tested for manifesting optimum yEGFP expression by monitoring the fluorescence intensities in minimal methanol histidine media supplemented with 0.5%, 1%, 2%, and 2.5% methanol. The pH of the media and the composition of the media (minimal or complex) also affect recombinant protein production. The effect of pH and the media composition was investigated in buffered media at pH 6 and unbuffered media of minimal methanol histidine media and methanol complex media. The results showed that the yEGFP expression increased up to the 5th day of induction and then decreased on the 6th day, 2% methanol showed the highest expression, and the pH and the media composition did not have any effect on the yEGFP expression. In conclusion, the optimum yEGFP expression can be obtained in 2% methanol on the 5th day of induction irrespective of minimal or complex media used.

Remodeling Bacillus amyloliquefaciens Cell Wall Rigidity to Reduce Cell Lysis and Increase the Yield of Heterologous Proteins.

Bacillus amyloliquefaciens has great potential as a host for heterologous protein production, but its severe autolytic behavior has precluded its industrial application to date. Because d,l-endopeptidase activity-guided cell wall rigidity is considered essential for autolysis resistance, we investigated the effects of d,l-endopeptidase genes lytE, lytF, cwlO, and cwlS play on the growth, lysis, and morphology remodeling of B. amyloliquefaciens strain TCCC11018. Individual and combinatorial deletion of lytE, lytF, and cwlS enhanced the cell growth and delayed cell lysis. For the best mutant with the lytF and cwlS double deletion, the viable cell number at 24 h increased by 11.90% and the cell wall thickness at 6 h increased by 25.87%. Transcriptomic and proteomic analyses indicated that the improvement was caused by enhanced peptidoglycan synthesis. With the lytF and cwlS double deletion, the extracellular green fluorescent protein and phospholipase D expression levels increased by 113 and 55.89%, respectively. This work broadens our understanding of the relationship between d,l-endopeptidases and B. amyloliquefaciens cell characteristics, which provides an effective strategy to improve the heterologous protein expression in B. amyloliquefaciens-based cell factories.

Development of a novel expression platform for heterologous protein production via deleting the p53-like regulator Vib1 in Trichoderma reesei

Trichoderma reesei is widely used as a protein production host due to its high natural capacity to secrete enzymes. Nonetheless, the complexity and abundance of secretome limit its extensive application in heterologous protein production. Here, a novel heterologous protein expression system with remarkable reduction of undesired background proteins was developed by deletion of the p53-like transcriptional factor Vib1. The vib1-deletion strain (Δvib1) exhibited a dramatic decrease in cellulase and protease secretion, whereas the growth of Δvib1 was comparable to that of the parental strain QM53, indicating that Δvib1 possesses a great potential for heterologous protein production. Therefore, the Aspergillus niger β-glucosidase-coding gene bglA was expressed in Δvib1 and QM53 to demonstrate the feasibility of Δvib1 as the protein production host. The bglA-expression strains QVB-1 (Δvib1:bglA) and Q53B-1 (QM53:bglA) produced approximately 17.2 IU/mg and 14.7 IU/mg of β-glucosidase activity, respectively. In addition, the β-glucosidase activity in the supernatant of QVB-1 remained constant after 4-week incubation whereas that of Q53B-1 decreased by more than 60%. Furthermore, transcription levels of the genes involved in the unfolded protein response were relatively decreased in Δvib1 compared with that in QM53, indicating the increased protein folding capacity of the endoplasmic reticulum in Δvib1. These results demonstrate the feasibility of using T. reesei Δvib1 as the host for heterologous protein production.

Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica.

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.

Coordinately express hemicellulolytic enzymes in Kluyveromycesmarxianus to improve the saccharification and ethanol production from corncobs

BackgroundHemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.ResultsTwo polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs.ConclusionsThe FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii

The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways.

Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

BackgroundMonoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.ResultsAdalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions.ConclusionThese results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.

Draft Genome Sequence of Paenibacillus polymyxa DSM 292, a Gram-Positive, Spore-Forming Soil Bacterium with High Biotechnological Potential.

Paenibacillus polymyxa DSM 292 was originally isolated from soil in 1947 due to its ability to produce antibiotics. The low proteolytic properties of strain DSM 292 warrant its examination as a host for heterologous protein production. Here, we report the draft genome sequence of DSM 292 as established by Illumina MiSeq paired-end sequencing.

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

BackgroundCyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca.ResultsTwo variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L−1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.ConclusionsWe have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves.

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized.

Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein

BackgroundThe Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and ^{13}hbox {C}-based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein.ResultsRNA-seq and ^{13}hbox {C}-based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion.ConclusionsTranscriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.

Engineering Yarrowia lipolytica for Use in Biotechnological Applications: A Review of Major Achievements and Recent Innovations.

Yarrowia lipolytica is an oleaginous saccharomycetous yeast with a long history of industrial use. It aroused interest several decades ago as host for heterologous protein production. Thanks to the development of numerous molecular and genetic tools, Y. lipolytica is now a recognized system for expressing heterologous genes and secreting the corresponding proteins of interest. As genomic and transcriptomic tools increased our basic knowledge on this yeast, we can now envision engineering its metabolic pathways for use as whole-cell factory in various bioconversion processes. Y. lipolytica is currently being developed as a workhorse for biotechnology, notably for single-cell oil production and upgrading of industrial wastes into valuable products. As it becomes more and more difficult to keep up with an ever-increasing literature on Y. lipolytica engineering technology, this article aims to provide basic and actualized knowledge on this research area. The most useful reviews on Y. lipolytica biology, use, and safety will be evoked, together with a resume of the engineering tools available in this yeast. This mini-review will then focus on recently developed tools and engineering strategies, with a particular emphasis on promoter tuning, metabolic pathways assembly, and genome editing technologies.

Comparison of Yeasts as Hosts for Recombinant Protein Production.

Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline.

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds 'Generally Recognised As Safe' status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains.

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018.

Reduction of Extracellular Proteases Increased Activity and Stability of Heterologous Protein in $${ Aspergillus}$$ A s p e r g i l l u s $${ niger}$$ n i g e r

The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT, was deleted in A. niger PY11 and the mutant (An\(\Delta \) prtT) characterised. Extracellular proteolytic activity of An\(\Delta \) prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate, under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An\(\Delta \) prtT harbouring the G. cingulata cutinase gene was increased 20- and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared using both strains was stable at \(4\,^{\circ }\hbox {C}\) for extended periods; however, during incubation at \(25^{\circ }\)C the heterologous cutinase in culture filtrates prepared using An\(\Delta \) prtT retained 80% activity after a two-week incubation versus the less than 3% activity that was retained in culture filtrates prepared using PY11. This indicates that the reduction of extracellular proteases greatly improves the stability of heterologous proteins produced by A. niger.

Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.