Patent Application
20230227879
The present invention relates to Actinomycetales strains for the improved formation of acarbose. Provided are Actinomycetales strains which are engineered to overexpress dTDP-D-glucose-4,6-dehydratase (AcbB) and/or uridyltransferase (GtaB). Also provided are Actinomycetales strains which are engineered to have a reduced or absent expression of the small carbohydrate binding protein (Cgt) and/or a reduced or absent expression of genes which are essential for carotenoid synthesis. Also provided are tools, methods and means to generate these strains.
Acarbose
The therapeutic agent acarviosyl-maltose (acarbose) is used since 1990 in the medical treatment of diabetes mellitus (Wehmeier and Piepersberg 2004; Wehmeier 2004). It shall support the rigorous diet plan of patients and prevent sugar peaks when consuming high carb meals. After oral application acarbose inhibits intestinal α-glucosidases, which leads to a retarded release of monosaccharides from starch- and sucrose-containing diets. By this, acarbose helps to control the rate of absorption of monosaccharides into the blood system and leads to a decreased postprandial blood and serum sugar level, which are assumed to be crucial in the context of cardiovascular disease mortality.
Acarbose is known and marketed in Europe and China as Glucobay (Bayer AG), in North America as Precose (Bayer Pharmaceuticals), and in Canada as Prandase (Bayer AG). Being an important and highly demanded drug for the treatment of diabetes mellitus, there is a need to provide acarbose in high yields and high quality. As the incidence of type II diabetes is continuously increasing worldwide, optimization of product yields and quality is an issue of current concern.
Acarbose Production Strains
Acarbose is naturally produced by different Actinomycetales, like Streptomyces coelicoflavus ZG0656 (Geng et al. 2009), Streptomyces glaucescens GLA.O (Rockser and Wehmeier 2009; Ortseifen et al. 2015) and Actinoplanes sp. SE50/110 (reviewed by Wehmeier and Piepersberg 2004), of which the latter is the wild type of industrial producer strains (Ortseifen 2016; Mahmud et al. 1999). The genus Actinoplanes was first introduced by Couch (1950) as member of the family Micromonosporaceae, order Actinomycetales, phylum Actinobacteria. Actinoplanes sp. SE50/110 (ATCC 31044, CBS 674.73) is a slow-growing natural derivate of SE50 (ATCC 31042, CBS 961.70) (Frommer et al. 1973). SE50 was isolated in 1970 during a screening program by the Bayer AG from a soil sample close to a coffee plantation in Kenia (Frommer et al. 1972). SE50/110 produces approximately 1 g·L1 acarbose, when maltose is provided in the medium (Wendler et al. 2014). Further production strains have been engineered, as described for example in (EP2601209B1) and (CN103298828B).
For Actinoplanes sp. SE50/110 it was shown, that the biosynthesis of acarviosyl-sugars depends on the supply of carbon sources in the culturing medium (Wendler et al. 2014). Growing on glucose, acarviosyl-glucose was formed as the major compound, whereas mainly acarviosyl-maltose was formed, when growing on maltose (Wendler et al. 2014), and acarviosyl-maltotriose, when growing on maltotriose (Ortseifen 2016).
Due to its medical and industrial relevance as wild type of industrial acarbose producer strains, Actinoplanes sp. SE50/110 was extensively studied in the last years: The complete genome (Schwientek et al. 2012), transcriptome (Schwientek et al. 2013) and proteome (Wendler et al. 2015b; Wendler et al. 2015a; Wendler et al. 2013) were analyzed comprehensively. This led to a refined genome sequence and an improved annotation in 2017 (GenBank: LT827010.1) (Wolf et al. 2017b). Also, an intergeneric conjugation system (Gren et al. 2016) as well as advanced genome editing tools by use of CRISPR/Cas9 (Wolf et al. 2016) were established, allowing targeted genetic engineering. Still, a reliable expression system enabling medium to strong gene expression is missing for Actinoplanes sp. SE50/110. Since a suitable system for the medium strong overexpression of singular genes did not exist before, different strategies were tested and evaluated according to the current invention, which led to the development of a novel expression system called pSETT4.
Acarbose Biosynthesis
The biosynthetic pathway of acarbose is based on monofunctional enzymes catalyzing single steps (
As the order of steps is unknown, they are shown in brackets. The first step of acarbose biosynthesis, a cyclic reaction by AcbC to form 2-epi-5-epi-valiolone from sedo-heptulose-7P, is missing in this illustration. Although becoming a focus of research in the last decades, still, the acarbose biosynthetic pathway is not completely unraveled yet. Only the first three steps of biosynthesis were experimentally confirmed. AcbC (ACSP50_3607), the first enzyme of the acarbose biosynthesis, catalyzes a cyclic reaction to generate 2-epi-5-epi-valiolone from sedo heptulose 7P7P (Stratmann et al., 1999). Phosphorylation to 2-epi-5-epi-valiolone-7P is catalyzed by the kinase AcbM (ACSP50_3603) in presence of ATP (Zhang et al. 2002) and epimerization to 5-epi-valiolone-7P by the cofactor independent epimerase AcbO (ACSP50_3606) (Zhang et al. 2002; Zhang et al. 2003).
The remaining steps of the model base on protein homologies and functional predictions (Zhang et al. (2002), Wehmeier (2003), Wehmeier and Piepersberg (2004), Wehmeier and Piepersberg (2009) and Wendler et al. (2013): NADH-dependent (polyol)dehydrogenase/reductase AcbL (ACSP50_3604) and the cyclitol dehydrogenase/oxidoreductase AcbN (ACSP50_3605) have been suggested to catalyze reduction and 5,6 dehydration to 1-epi-valienol-7P. Phosphorylation to 1,7-diphospho-1-epi-valienol is assumed to be catalyzed by the 1-epi-valienol-7-phosphate-1-kinase AcbU (ACSP50_3595) and/or hydrolase AcbJ (ACSP50_3600). Nucleotidylation to NDP-1-epi-valienol-7P is possibly catalyzed by a GlgC-related NDP-polyol synthase AcbR (ACSP50_3597) (1-epi-valienol-1,7-bisphosphate-1-adenylyltransferase), and transfer of the activated intermediate to an activated amino sugar seems to be mediated by the glycosyltransferases Acbl (ACSP50_3599) and/or AcbS (ACSP50_3596) to generate acarviosine-7P.
The activated amino sugar is supposed to be synthesized from D-glucose-1-phosphate in three steps (Wehmeier and Piepersberg 2004; Wehmeier and Piepersberg 2009; Zhang et al. 2002), which are: (i) nucleotidylation by the dTDP-glucose-synthase AcbA (ACSP50_3609) to dTDP-D-glucose, (ii) dehydration by dTDP-D-glucose-4,6-dehydratase AcbB (ACSP50_3608) to dTDP-4-keto-6-deoxy-D-glucose, and (iii) amination by a GabT-like aminotransferase AcbV (ACSP50_3594) to dTDP-4-amino-4,6-dideoxy-D-glucose (Diaz-Guardamino Uribe 2000; Zhang et al. 2019).
Glucose-1P is a branching metabolite displaying an important role in different pathways, like the glycogen metabolism, the galactose metabolism and—after conversion to glucose-6P—the glycolysis (Frey 1996; Purves 2006). UDP-glucose-1P uridyltransferase GtaB catalyzes the conversion of glucose-1P and UDP-glucose into each other.
Last, maltose is transferred in a one-step reaction (Hemker et al. 2001), potentially by AcbS. However Acbl or AcbJ have also been proposed to catalyze the transfer reaction (Wehmeier and Piepersberg 2004; Wendler et al. 2013). Another candidate for this reaction might be the amylomaltase AcbQ (ACSP50_3601).
In Actinoplanes sp. SE50/110, the biosynthesis genes are organized in the acarbose biosynthesis gene cluster (acb gene cluster), which was first identified in 1999 by Stratmann et al. and subsequently sequenced (GenBank: Y18523.4) (Stratmann et al. 1999; Thomas 2001). The cluster contains 22 genes (
Beside of the already mentioned biosynthetic genes (acbCMOLNUJRSIVBA), the cluster encodes functions in extracellular starch degradation (AcbEZ, ACSP50_3610 and ACSP50_3590), transglycosylation (AcbD, ACSP50_3611) and in export of acarbose (AcbWXY, ACSP50_3591-3). Besides, an acarbose-7-kinase (AcbK, ACSP50_3602) and an intracellular amylomaltase (AcbQ) are encoded, which were assigned to a function within the carbophore (Wendler et al. 2015b; Schwientek et al. 2012; Wehmeier and Piepersberg 2009). The function of AcbP (ACSP50_3598), annotated as NTP-pyrophosphohydrolase, is unknown.
Actinoplanes Proteins with Possible Metabolic Relevance
The singular CBM-20 domain protein Cgt is one of the most strongly expressed genes in Actinoplanes sp. SE50/110 and in derived acarbose producer strains (Ortseifen 2016; Wendler et al. 2015a; Schwientek et al. 2013). It is secreted via the Sec-pathway according to SignalP-analysis (Almagro Armenteros et al. 2019) and makes up to 8% of the total secreted proteome of this organisms (data not shown). Cgt contains 149 amino acids and a CBM-20-domain of fold-family 1, functional group A, characterized by a p-sandwich structure (Schwientek et al. 2013; Guillén et al. 2010). Members of this family are described to bind starch (Guillén et al. 2010).
Methods for Gene Deletion in Actinoplanes
The establishment of an intergeneric conjugation system (Gren et al. 2016) and the CRISPR/Cas9 technique (Wolf et al. 2016), allow genome editing in Actinoplanes sp. SE50/110. In addition, according to the current invention the inventors have successfully established a novel deletion system by homologous recombination, which uses an integrase-free vector backbone and CodA for counter selection, like described by Zhao et al. (2017). By this, the genetic toolbox for Actinoplanes sp. SE50/110 could be further extended. As proof of principle the novel deletion system was successfully tested for deletion of the example gene cgt. Homologous recombination (HR) is a common process in Actinobacteria, which can be technically used to create deletion mutants by double crossover. Temperature-sensitive replicons, like the pSG5 replicon, can support and force this process. (Du et al. 2015; Garg and Parry 2010; Myronovskyy et al. 2009; Zhang and Parry 2007). Further methods exist in the art, e.g. CRISPR-Base Editing System for the exchange of single nucleotides, CRISPR-BEST according to Tong et al. 2019, CRISPRi/dCas9 according to Qi et al. 2013, RNA interference etc.
Methods for Gene Overexpression in Actinoplanes
Actinoplanes sp. SE50/110 has been extensively studied in the last decades. Appropriate expression systems are difficult to design, see Schaffert et al. (2019). The whole content of the publication and in particular the description of the expression systems and promoters for the genetic manipulation of Actinoplanes are included herein in their entirety.
Previous studies have shown successful expression of genes by use of pKC1139 in A. teichomyceticus (Horbal et al. 2012). However, the replicative pSG5-based vector pKC1139 (constructed by Bierman et al. (1992)) turned out to be unsuitable for expression of homologous genes in Actinoplanes sp. SE50/110, as unwanted vector integration by homologous recombination occurs, see Schaffert et al. (2019). This seems to be a favored process, putatively due to the high metabolic costs of vector replication. Without being bound by theory, a protein encoded by ACSP50_7170 in SE50/110, predicted as recombinase A (recA) might catalyze the recombination process. Interestingly, no homologue of recA was found in the genome of A. teichomyceticus. Presence of recA in a. sp. SE50/110 and lack in A. teichomyceticus provides a conclusive explanation, why HR-mediated vector-integration has not been reported for A. teichomyceticus before. A pSG5-based replicative expression system may therefore be implemented by deletion of the recombinase gene recA in Actinoplanes sp. SE50/110.
Other replicative Streptomyces-E. coli shuttle plasmids, like pKC1218, which is based on the SCP2*-replicon (Kieser et al. 2000), and pSOK101, which is based on the plJ101-replicon (Zotchev et al. 2000), did not give exconjugants with Actinoplanes sp. SE50/110 (Gren 2017). These replicons are probably unstable or inactive in SE50/110, which is in accordance with findings from the related species A. teichomyceticus (Horbal et al. 2012).
By use of integrative vector systems, a genetic duplication can be achieved by integration of the complete vector carrying an additional gene copy at a distinct genomic location. This process is mediated by phage integrases. Phage integrases catalyze the targeted and unidirectional recombination of two attachment sites: attP, which is localized on the plasmid, and attB, which is localized in the host chromosome (te Poele et al., 2008). After integration, the vector is flanked by the attachment site left (attL) and right (attR), which are derived from attP-attB-recombination (te Poele et al., 2008).
Four different integrative vectors have been described for Actinoplanes sp. SE50/110 (Gren et al. 2016): Two are based on the integration mechanism of the phage φC31 (pSET152 and plJ6902). The vectors pRT801/2 and pSOK804 are based on the integration mechanism of the phage φBT1 and of the VWB-phage. However, doubling of relative transcript amounts by use of the native promoters was not achieved, see Schaffert et al. (2019).
Evaluation of Homologous and Heterologous Promoters for Integrative Vectors
A method to evaluate homologous and heterologous promoters with regard to their strength is provided in Schaffert et al. (2019), which is incorporated herein in its entirety. In brief, the integrative φC31-based vector pSET152 was used for promoter screening in Actinoplanes sp. SE50/110 (Gren et al. 2016). The promoter strengths of 13 homologous and heterologous promoters were analyzed on protein level, and 12 of these were analyzed on transcript level (Table 1,
Strategy
For the current invention, the acarviosyl-maltose metabolism was studied by gene deletion and overexpression, leading to a set of associated tools and methods to engineer strains for the improved production of acarbose. In order to improve the acarviose-synthesis, three different strategies were followed: (i) increasing of the gene dose of acb genes to enhance the flux through the acarbose biosynthesis, (ii) deployment of precursors of acarbose biosynthesis and (iii) reducing the metabolic burden (
Furthermore, growth experiments of the wild type and a regulator mutant ΔmerR exposed to and hidden from light were conducted, revealing a negative influence of light-induced stress and carotenoid formation on the acarbose production. Consequently, the acarbose production can furthermore be improved by reducing the carotenoid formation.
A. Methylerythritolphosphate (MEP) pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which is also known as alternative metabolic pathway of the mevalonate pathway.
B-C: Carotenogenesis. B. Formation of Lycopene from isoprenoid precursors. C. Synthesis of the glycosylated carotenoid Sioxanthin in Salinospora tropica CNB-440 (
D. Genomic organization of the identified genes in Actinoplanes sp. SE50/110. Gene cluster 2b displays homologies to the sioxanthin gene cluster from Salinospora tropica CNB-440 according to analysis by antiSMASH, a rapid genome-wide identification tool for the annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genomes (Weber et al., 2015).
D. Significant differences compared to the empty vector control were observed for the normalized peak areas of UDP-glucose (p-values of a two-sided t-test: Ptip: 0.01068, Pgap: 0.001356) and of the mass m/z=545 [M-H+] (p-value of a two-sided t-test: Ptip: 0.0412).
The Sequence Listing associated with this application is filed in electronic format and hereby incorporated by reference into the specification in its entirety.
Unless otherwise defined, all scientific and technical terms used in the description, Figures and claims have their ordinary meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control. The materials, methods, and examples are illustrative only and not intended to be limiting. Unless stated otherwise, the following terms used in this document, including the description and claims, have the definitions given below.
The terms “comprising”, “including”, “containing”, “having” etc. shall be read expansively or open-ended and without limitation. Singular forms such as “a”, “an” or “the” include plural references unless the context clearly indicates otherwise. Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. The terms “at least one” and “at least one of” include for example, one, two, three, four, five, six, seven, eight, nine, ten or more elements.
It is furthermore understood that slight variations above and below a stated range can be used to achieve substantially the same results as a value within the range. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.
Where protein or amino acid sequences are provided throughout the application it is also understood by the skilled person that single or multiple amino acids may be exchanged by amino acids with similar properties to achieve substantially the same effect, i.e. an equivalent result.
The skilled person furthermore knows that a defined protein or amino acid sequence may be encoded by various nucleic acid sequences. For a given amino acid sequence as defined herein, each of the countable nucleic acid sequences encoding the specific amino acid sequence shall be deemed to be disclosed herein. Where nucleic acid sequences are provided throughout the application it is furthermore understood that silent mutations may be introduced.
O-{4,6-dideoxy-4[1S-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino-α-D-gluco-pyranosyl}-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose or “acarbose” is a cyclitol-containing aminoglycoside, composed of a pseudodisaccharide and an α-1,4-glycosidic bound maltose (Wehmeier and Piepersberg 2009). The pseudodisaccharide, named acarviose, is built by an unsaturated C7-aminocyclitol, also referred as valienol or valienamine, which is connected to C4 of a 4,6-didesoxy-D-glucose by a nitrogen bond (cf.
“Overexpression” of a gene product or protein as described herein refers to an increase in expression compared to the wild type or a specified reference strain. Preferably, the reference strain or control is the strain which has not been engineered for the specific overexpression of the respective gene(s) or protein(s). For example, the control does not comprise a vector comprising an expression cassette for the respective gene product or protein. For example, the overexpression of the gene product may be an increase during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
Preferably, overexpression is an increase of the gene product or protein by a factor of at least 1.5 or at least a factor of 2 compared to the control. With regard to transcript amounts and if not defined otherwise herein, strong overexpression refers to a log 2(fold change) >6. With regard to transcript amounts and if not defined otherwise herein, weak overexpression refers to a log 2(fold change)<2. With regard to transcript amounts and if not defined otherwise herein, medium strong overexpression refers to a log 2(fold change) ≥2 and ≤6.
The expression of a gene product or protein as described herein is “absent or reduced” if the respective gene has been deleted or mutated in such a way that its gene product is not expressed at all or in a significantly decreased amount (e.g. less than 0.75 fold or less than 0.5 fold). The expression of a gene product or protein as described herein is also considered absent or reduced if the gene product or protein has lost functionality, e.g. in a transient or permanent way, e.g. by mutation or knockdown. Methods to monitor the amount and or activity of a gene product or protein are known in the art and are also described herein in an exemplary way. In general, suitable methods to obtain an absent or reduced expression of a gene product are methods that alter the genetic sequence or elements of gene expression (e.g. by deletion or point mutations) and/or methods that negatively affect the transcription and translation of a gene or the activity or half-life of the gene product (protein).
If not specified otherwise the symbol “Δ” refers to a “deletion mutant”, i.e. a mutant wherein a specific gene sequence bas been at least partially deleted.
The “early growth phase” is the time, in which the Actinoplanes strain adapts to the medium and in which the cell dry weight is below 3 g·L1. After adaption to the environment, the culture metabolizes the nutrients supplied by the medium and starts to grow. Since Actinoplanes is growing in a spherical mycelium, which can only expand to the outside of the sphere, the cells in the middle are shielded from nutrients and have only limited space for cell division. Therefore, only the cell in the outer layer of the spherical mycelium are dividing. By this, growth of Actinoplanes is linear and not exponential—in contrast to other bacteria, which are growing unicellular. The growth phase is called “linear growth phase” for Actinoplanes ssp. and starts at a cell dry weight of 3 g·L−1. The “stationary phase” is defined as growth phase, in which the cells reach the capacity limits (of space and nutrients) respectively in which growth decreases due to the formation of inhibitory by-products or other chemical and physical factors such as changes in the osmolarity or pH. The stationary phase is the growth phase, in which the number of dying cells equals the number of dividing cells. This phase usually starts at a cell dry weight of 16-18 g·L−1 in maltose minimal medium.
The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating a nucleic acid molecule to which it is linked.
The term “expression cassette”, as used herein, refers to a nucleic acid molecule comprising at least a gene for expression and a regulatory sequence, such as a promoter.
A “promoter” is a nucleic acid sequence which leads to initiation of transcription of a particular gene.
A “strong promoter” as defined herein is a promoter, which leads to a normalized glucuronidase activity of at least 5·10−4 [L·g−1·min−1] in the glucuronidase assay, and/or which leads to a 350-fold relative transcription (in log 2(fold change)) of the gusA gene compared to the promoterless pGUS control vector. A detailed description of a method for characterizing the strength of a promoter is provided within the examples and in (Schaffert, et al. 2019).
Examples include the promoters of
A “medium strong promoter” is defined as promoter, which leads to a normalized glucuronidase activity of at least 1·10−4 [L·g−1·min−1] was achieved in the glucuronidase assay, and/or which leads to a 10-fold relative transcription (in log 2(fold change)) of the gusA gene compared to the promoterless pGUS control vector. Examples include the promoters of
In some cases, the medium strong promoter leads to a normalized glucuronidase activity of at least 1·10−4 [L·g−1·min−1] and maximal 5·10−4 [L·g−1·min−1] in the glucuronidase assay.
A “weak promotor” is defined as promoter, which leads to a normalized glucuronidase activity of below 1·10−4 [L·g−1·min−1], and/or which leads to a relative transcription of below 10-fold compared to the promoterless pGUS control vector.
The term “Cgt” (ACSP50_5024, previously: ACPL_5091) refers to extracellular small carbohydrate binding protein, previously described as cyclomaltodextrin glucanotransferase due the high similarity to the C-terminal domain of cyclodextrin glycosyltransferases, obtained from Actinoplanes sp., e.g. strain ATCC 31044/CBS 674.73/SE50/110. Cgt protein is encoded by the gene cgt. Sequence(s) are described herein (SEQ ID No. 20) or are accessible via UniProt Identifier G8S155 (G8S155_ACTS5). Different isoforms and variants may exist for the different strains and are all comprised by the term. Where a specific mutation can be exchanged without changing the described catalytic properties of the initial sequence, it is clear that the sequence having such a functionally silent mutation is equivalent with regard to the initial sequence. In addition, the protein may furthermore be subject to various modifications, e.g, synthetic or naturally occurring modifications.
The term “AcbB” (ACSP50_3608, previously ACPL_3681) refers to dTDP-D-glucose-4,6-dehydratase obtained from Actinoplanes sp., e.g. strain ATCC 31044/CBS 674.73/SE50/110, which is probably involved in the biosynthesis of the acarviose moiety of acarbose. AcbB protein is encoded by the gene acbB. Sequence(s) are described herein (SEQ ID No. 13) or are accessible via UniProt Identifier Q9ZAE8 (RMLB_ACTS5). Different isoforms and variants may exist for the different strains and are all comprised by the term. Where a specific mutation can be exchanged without changing the described catalytic properties of the initial sequence, it is clear, that the sequence having such a functionally silent mutation is equivalent with regard to the initial sequence. In addition, the protein may furthermore be subject to various modifications, e.g, synthetic or naturally occurring modifications.
The term “GtaB” also “GaIU” (ACSP50_7820, previously ACPL_7811) refers to UTP-glucose-1-phosphate uridylyltransferase obtained from Actinoplanes sp., e.g. strain ATCC 31044/CBS 674.73/SE50/110. GtaB seems to catalyze the conversion of glucose-1P and UDP-glucose into each other and might be involved in the precursor supply for acarbose. GtaB protein is encoded by the gene gtaB. Sequence(s) are described herein (SEQ ID No. 19) or are accessible via UniProt Identifier G8S608 (ACPL_7811). Different isoforms and variants may exist for the different strains and are all comprised by the term. Where a specific mutation can be exchanged without changing the described catalytic properties of the initial sequence, it is clear that the sequence having such a functionally silent mutation is equivalent with regard to the initial sequence. In addition, the protein may furthermore be subject to various modifications, e.g, synthetic or naturally occurring modifications.
As defined herein, a “gene which is essential for carotenoid synthesis” is defined as a gene which is positively required for the synthesis of a carotenoid. Actinoplanes are known to produce a variety of soluble pigments including yellow, orange and pink pigments of the class carotenoids. In Actinoplanes, the set of genes which are essential for carotenoid synthesis include genes from the MEP/DOXP pathway, genes of terpene cluster 1, genes of terpene cluster 2a, genes of terpene cluster 2b and genes of camphene-like monoterpene biosynthesis terpene cluster 3. Genes of the MEP/DOXP pathway comprise
Genes of terpene cluster 1 comprise
Genes of terpene cluster 2a comprise
Genes of terpene cluster 2b comprise
Another gene which is essential for carotenoid synthesis is polyprenyl synthetase gene crtE (ACSP50_3873, SEQ ID No. 49).
Genes of camphene-like monoterpene biosynthesis terpene cluster 3 comprise
While Actinomycetales strain Actinoplanes sp. SE50/110 was used as a model strain for the current invention, it is clear for the skilled person, that the general mechanisms and findings can be applied for other acarbose producing strains such as those strains which are currently used for the commercial production of acarbose. According to some embodiments, the Actinomycetales strain is a Micromonosporaceae strain. According to some embodiments, the Actinomycetales strain is an Actinoplanes strain. According to some embodiments, the Actinomycetales strain is Actinoplanes SE50 (ATCC 31042, CBS 961.70) (Frommer et al. 1973), Actinoplanes sp. SE50/110 (ATCC 31044, CBS 674.73) or an Actinoplanes strain derived thereof. In some embodiments, the Actinomycetales strain is an Actinoplanes strain which is commercially used for acarbose production. In some embodiments, the Actinomycetales strain is an Actinoplanes strain which is commercially used for Acarbose production, such as SN223-29-47, C445-P47, SN12755-38, SC3687-18-43, SC7177-40-17 or SN19910-37-21 as disclosed e.g. in EP 2601209 B1 and CN103298828 B, or a strain derived thereof.
Improvement of acarbose production refers to an increase in yield of acarbose over a specific time (either in total or relative to cell growth) and/or improvement of the purity of the acarbose, e.g. the decrease of side-products and/or acarbose analogs such as component C. Cultivation of the Actinoplanes strain can occur as known in the art or as described herein. In some embodiments, cultivation of the Actinoplanes strain occurs in maltose minimal medium.
According to a first aspect of the current invention, there is provided a method to engineer an Actinomycetales strain, such as an Actinoplanes strain, for the improved production of acarbose. According to some first embodiments according to the first aspect, the method according to the first aspect comprises engineering the Actinomycetales strain for absent or reduced expression of extracellular small carbohydrate binding protein Cgt (SEQ ID No. 20).
Surprisingly, deletion of carbohydrate binding protein Cgt (SEQ ID No. 20) resulted in an improved production of acarbose. An increase of the final acarbose yield between 8.3 and 16.6% was achieved in three independent shake flask cultivations (cf. example “Δcgt displays improved acarbose formation on maltose minimal medium”,
Furthermore, in comparison with the wildtype, the gene deletion mutant Δcgt displayed no apparent growth phenotype in screening experiments testing for different carbon sources, or under carbon-limited conditions (cf. examples “Analysis of cgt expression during growth on different carbon sources”, “Δcgt on different carbon sources or under carbon-limited conditions”,
Without being bound by theory, Cgt was found to be highly expressed in Actinoplanes sp. SE50/110 according to comprehensive studies of the extracellular proteome (Wendler et al. 2013; Ortseifen 2016) and transcriptome (Schwientek et al. 2013). Its gene product is exported into the extracellular space making up for about 8% of the whole secreted proteome. The inventors have analyzed the distribution of CBM-20 single-domain proteins in the prokaryotic world by BlastP analysis. Interestingly, singular CBM-20 domain-proteins were found in only 17 other species (cf. example “Distribution of single-domain CBM-20 proteins in the eubacterial world”). Most of these are found in species of the order Actinomycetales, for example in all strains of the genus Actinoplanes. Without being bound by theory, by deletion or reduced expression of cgt, energy and resources, such as ATP and amino acids, are relieved.
These resources may then be redirected to the acarbose biosynthesis, which is a growth-associated product.
According to some embodiments according to the first aspect, the method comprises deletion or mutation of the gene encoding extracellular small carbohydrate binding protein Cgt (SEQ ID No. 20). The establishment of an intergeneric conjugation system (Gren et al. 2016) and the CRISPR/Cas9 technique (Wolf et al. 2016), allows genome editing in Actinoplanes sp. SE50/110. In some embodiments according to the first aspect engineering the Actinomycetales strain for absent or reduced expression may occur using CRISPR/Cas9 technique. In some embodiments, engineering the Actinomycetales strain for absent or reduced expression may occur as described by (Wolf et al. 2016). In some embodiments engineering the Actinomycetales strain for absent or reduced expression may occur as described herein, e.g. as described in the example “Deletion of the gene cgt by CRISPR/Cas9 technique” or “Deletion system based on homologous recombination and counterselection with the cytosine deaminase CodA”.
For example, the inventors have successfully established a novel deletion system by homologous recombination, which uses an integrase-free vector backbone and CodA for counter selection, like described by Zhao et al. (2017).
According to some second embodiments according to the first aspect, the method according to the first aspect comprises engineering the Actinomycetales strain for absent or reduced expression of at least one gene which is essential for carotenoid synthesis. In some embodiments, the carotenoid is the orange pigment of Actinoplanes or a derivative thereof. In some different or the same embodiments, the carotenoid is a C40-carotenoid.
Engineering the Actinomycetales strain for absent or reduced expression may occur as described previously for the current aspect. According to some embodiments according to the first aspect, the method comprises deletion or mutation of the gene which is essential for carotenoid synthesis.
Actinoplanes are known to produce a variety of soluble pigments including yellow, orange and pink pigments of the class carotenoids (Parenti and Coronelli 1979). The inventors observed, that strong pigmentation was associated with acarbose production losses. This was confirmed by comparing growth and acarbose yields of cultures exposed to and covered from light (cf. example “Light-dependent carotenoid-formation and oxidative stress reduce acarbose production in Actinoplanes sp. SE50/110”,
From these findings it is not only plausible that the produced pigments are not essential (e.g. in a technical setup for commercial acarbose production) but also that reducing or depleting the carotenoid synthesis in Actinoplanes can be used to improve the acarbose formation. To this end, the method according to the first aspect comprises reducing or depleting the expression of at least one gene which is essential for carotenoid synthesis.
The inventors could furthermore reconstruct the carotenogenesis in Actinoplanes sp. SE50/110 (cf. example “Analysis of the functional relevance of carotenoid formation”,
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is a gene of the MEP/DOXP pathway, such as
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is a gene of terpene cluster 1, such as
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is zeta-phytoene desaturase gene crtl (ACSP50_0147, SEQ ID No. 10). As discussed before, carotenoid formation is dispensable under laboratory conditions. In order to improve acarbose production, switching off the concurring carotenoid biosynthesis pathway, in particular by deletion of the central gene crtl, can be used for strain development.
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2a, such as
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2b, such as
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is polyprenyl synthetase gene crtE (ACSP50_3873, SEQ ID No. 49).
According to some embodiments according to the current aspect and embodiments, the at least one gene essential for carotenoid synthesis is a gene of camphene-like monoterpene biosynthesis terpene cluster 3, such as
Since carotenoids influence the fluidity of membranes, lack of carotenoids and in particular of the C40-carotenoid can also affect the surface and mycelial structure of Actinoplanes sp. SE50/110. With regard to production break-up of mycelial lumps is advantageous to increase the mycelial surface and the number of biochemically available cells.
According to some further embodiments, the method according to the first aspect comprises engineering the Actinomycetales strain for overexpression of MerR-/HTH-transcriptional regulator gene merR (ACSP50_0145, SEQ ID No. 11). Engineering the Actinomycetales strain for overexpression may occur as described elsewhere herein.
Beside the mentioned genes which are essential for carotenoid synthesis, the inventors surprisingly identified a transcriptional repressor for the carotenoid synthesis among the genes of terpene cluster 1: ACSP50_0145 (SEQ ID No. 11, MerR-/HTH-transcriptional regulator gene merR) cf. example “Deletion of merR in SE50/110 induces carotenoid formation without exposure to light”,
Consistent with this, the acarbose production was found to be decreased. When illuminated, both wild type and AmerR are strongly pigmented and the final acarbose concentrations were similar for both strains, reaching approx. 0.52 g·L−1 (
Under dark conditions, AmerR produces approx. 15% less acarbose than the wild type (0.70 g·L−1) (
According to some third embodiments according to the first aspect the method comprises engineering the Actinomycetales strain for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13).
According to the current invention it was surprisingly found that overexpression of the acb gene encoding the dTDP-D-glucose-4,6-dehydratase AcbB increased the final acarbose concentration significantly by approx. 50%. This was particularly surprising, because other genes of the Acb cluster such as AcbC did not lead to an improved formation of acarbose.
Furthermore, the observed increase was superior compared to the observed increase for overexpression of the complete Acb cluster as described by Zhao et al. (Zhao, Xie, et al. 2017).
According to some embodiments, the strain does not comprise engineering the Actinomycetales strain for overexpression of other genes of the Acb cluster, except for AcbA.
The dTDP-D-glucose-4,6-dehydratase AcbB seems to be involved in the generation of an activated amino sugar from D-glucose-1P which is a feeding pathway of the acarbose biosynthesis (
Overexpression of AcbB as described herein refers to an increase in expression for AcbB compared to the wild type or a specified reference strain/control. For example, the overexpression of the gene product may be an increase during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
Preferably, as described herein, overexpression of AcbB refers to an increase of AcbB transcript and/or protein by a factor of at least 1.5 or at least a factor of 2 compared to the control. With regard to AcbB transcript amounts, and if not defined otherwise herein, strong overexpression refers to a log 2(fold change) >6. With regard to AcbB transcript amounts and if not defined otherwise herein, medium strong overexpression refers to a log 2(fold change) ≥2 and ≤6.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a factor of a log 2(fold change) of at least 1.5 or at least 2, during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) ≥2 and ≤6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time, such as during the early growth phase and/or during the linear growth phase.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) >6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
In overexpression mutants with expression vectors comprising heterologous promoters the relative transcription of acbB decelerated from 4.06- to 3.33-fold (log 2(fold change)) between the two sampling times in pSETT4tip::acbB (medium strong promoter) and from 6.54- to 2.05-fold in in pSETT4gap::acbB (strong promoter) (cf. example “Medium overexpression of acbB leads to improved acarbose formation”).
According to some embodiments, engineering the Actinomycetales strain for overexpression of a gene according to the first aspect may occur by any method known in the art or described herein.
As described within the example “Medium overexpression of acbB leads to improved acarbose formation”, two pSETT4-based overexpression mutants were created, in which acbB is transcribed under control of the medium strong tipA-promoter or the strong gapDH-promoter.
The native promoter was used in both the pSET152- and the pSETT4-vector background as control. In particular the mutant with acbB transcribed under control of the heterologous tipA-promoter displayed enhanced acarbose production compared to the control strains (
According to some embodiments, engineering the Actinomycetales strain for overexpression of a gene according to the first aspect may occur by introducing a vector comprising an expression cassette for AcbB (SEQ ID No. 13) into the Actinomycetales strain. In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. A vector is derived from another vector, if it comprises at least one, two, three, four elements of the second vector.
According to some embodiments, engineering the Actinomycetales strain for overexpression of a gene according to the first aspect may occur by introducing a vector comprising an expression cassette for AcbB (SEQ ID No. 13) into the Actinomycetales strain. In some of these or other embodiments the expression cassette is under the control of a medium strong promoter, as characterized by a normalized glucuronidase activity of at least 1x·10−4, preferably between 1x·10−4 and 5×10−4 [L·g−1·min−1] in a glucuronidase assay, e.g. as described elsewhere herein. In some embodiments said promoter is selected from efp promoter (SEQ ID No. 92), cdaR promoter (SEQ ID No. 97), rpsL promoter (SEQ ID No. 99), rpsJ promoter (SEQ ID No. 93), cgt promoter (SEQ ID No. 91), or tipA promoter (SEQ ID No. 81). In some embodiments the promoter is the tipA promoter (SEQ ID No. 81). Excellent results for acarbose production were obtained with pSETT4tip::acbB, cf.
In some embodiments the expression cassette is under the control of a strong promoter, as characterized by a normalized glucuronidase activity of at least 5×10−5 [L·g−1·min−1] in a glucuronidase assay, e.g. as described elsewhere herein. In some embodiments said promoter is selected from apm promoter (SEQ ID No. 96), ermE* promoter (SEQ ID No. 98), katE promoter (SEQ ID No. 94), moeE5 promoter (SEQ ID No. 95) or gapDH promoter (SEQ ID No. 82).
According to some embodiments, the method according to the first aspect comprises engineering the Actinomycetales strain for medium overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) and optionally AcbA (SEQ ID No. 12). In some embodiments, which are also compatible with all other embodiments described herein if not explicitly stated otherwise, genetic engineering does not result in an increase of transcript and/or protein by a log 2(fold change) ≥2 for Acb genes other than AcbB and AcbA. In some of embodiments which are also compatible with all other embodiments described herein, genetic engineering does not result in an increase of transcript and/or protein by a log 2(fold change) ≥2 for AcbC.
Upon overexpression of AcbB, further genes of the acb gene cluster were not significantly affected, e.g. in the early growth phase, like shown for acbA and acbV (
According to some embodiments, the method according to the first aspect comprises engineering the Actinomycetales strain for overexpression of AcbB (SEQ ID No. 13) and AcbS (ACSP50_3596) and/or Acbl (ACSP50_3599).
By (additional) overexpression of AcbS and/or Acbl, the transfer reaction of the amino sugar to the cyclitol precursor can be strengthened. According to the current model (see
According to some embodiments, the method according to the first aspect comprises engineering the Actinomycetales strain for overexpression of AcbB (SEQ ID No. 13) and AcbCUJ (AcbC (ACSP50_3607) and/or AcbU (ACSP50_3595) and/or AcbJ (ACSP50_3600)) and/or AcbSI (AcbS (ACSP50_3596) and/or Acbl (ACSP50_3599)). Without being bound by theory, this combination can plausibly reinforce both acarbose synthesis strands.
According to some fourth embodiments according to the first aspect, the method comprises engineering the Actinomycetales strain for overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID No. 19).
By medium overexpression of gtaB, an increase of 8.5% of the final acarbose concentration was observed, cf. example “Medium overexpression of gtaB leads to improved acarbose formation”,
Overexpression of GtaB (SEQ ID No. 19) as described herein refers to an increase in expression for GtaB transcript and/or protein compared to the wild type or a specified reference strain/control. For example, the overexpression of the gene product may be an increase during the early growth phase and/or during the linear growth phase and/or during the stationary phase, and/or an increase during any other time.
Preferably, overexpression is an increase of GtaB transcript and/or protein by a factor of at least 1.5 or at least a factor of 2 compared to the control. With regard to GtaB transcript amounts and if not defined otherwise herein, strong overexpression refers to a log 2(fold change) >6. With regard to GtaB transcript amounts and if not defined otherwise herein, medium strong overexpression refers to a log 2(fold change) ≥2 and ≤6.
According to some embodiments the overexpression of UDP-glucose-1P uridyltransferase GtaB is the increase of the expression of GtaB by a factor of a log 2(fold change) of at least 1.5 or at least 2 during the early growth phase and/or during the linear growth phase and/or during the stationary phase, and/or an increase during any other time.
In one of the overexpression mutants described herein, the relative transcript amount of the gene gtaB is 2.64-fold increased (log 2(fold change)) (
According to some embodiments the overexpression of UDP-glucose-1P uridyltransferase GtaB is the increase of the expression of GtaB transcript and/or protein by a log 2(fold change) ≥2 and ≤6 during the early growth phase and/or during the linear growth phase and/or during the stationary phase, and/or an increase during any other time. According to some embodiments the overexpression of UDP-glucose-1P uridyltransferase GtaB is the increase of the expression of GtaB by a log 2(fold change) ≥3 and ≤5 during the early growth phase and/or during the linear growth phase and/or during the stationary phase, and/or an increase during any other time.
According to some embodiments the overexpression of UDP-glucose-1P uridyltransferase GtaB is the increase of the expression of GtaB by a log 2(fold change) ≥6 during the early growth phase and/or during the linear growth phase and/or during the stationary phase.
According to some embodiments, engineering the Actinomycetales strain for overexpression of a gene according to the first aspect may occur by introducing a vector comprising an expression cassette for GtaB (SEQ ID No. 19) into the Actinomycetales strain. In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. A vector is derived from another vector, if it comprises at least one, two, three, four elements of the second vector.
According to some embodiments, engineering the Actinomycetales strain for overexpression of a gene according to the first aspect may occur by introducing a vector comprising an expression cassette for GtaB (SEQ ID No. 19) into the Actinomycetales strain.
In some of these or other embodiments the expression cassette is under the control of a medium strong promoter, as characterized by a normalized glucuronidase activity of between 1x·10−4 and 5×10−5 [L·g−1·min−1] in a glucuronidase assay, e.g. as described elsewhere herein. In some embodiments said promoter is selected from efp promoter (SEQ ID No. 92), cdaR promoter (SEQ ID No. 97), rpsL promoter (SEQ ID No. 99), rpsJ promoter (SEQ ID No. 93), cgt promoter (SEQ ID No. 91), or tipA promoter (SEQ ID No. 81). In some embodiments the promoter is the tipA promoter (SEQ ID No. 81). Good results for acarbose production were obtained for example with pSETT4tip::gtaB, cf.
In some embodiments the expression cassette is under the control of a strong promoter, as characterized by a normalized glucuronidase activity of at least 5×10−5 [L·g−1·min−1] in a glucuronidase assay, e.g. as described elsewhere herein. In some embodiments said promoter is selected from apm promoter (SEQ ID No. 96), ermE* promoter (SEQ ID No. 98), katE promoter (SEQ ID No. 94), moeE5 promoter (SEQ ID No. 95) or gapDH promoter (SEQ ID No. 82).
According to some further or the same embodiments of the first aspect, the method comprises engineering the Actinomycetales strain for medium overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) and GtaB (SEQ ID No. 19).
It was surprisingly found that overexpression of GtaB triggers improved acarbose formation. By medium overexpression of acbB (e.g. by use of the tipA-promoter), a positive effect on acarbose production was observed yielding approx. 50% more acarbose in two independent cultivations.
Therefore, the improvement of the acarbose biosynthesis by overexpression of singular acb gene AcbB was achieved. Furthermore, by medium overexpression of gtaB, an increase of 8.5% of the final acarbose concentration was observed. It is plausible, that by a combined overexpression of acbB and gtaB, the flux through the amino sugar biosynthesis is improved leading to a further enhancement of acarbose production.
Without being bound by theory, strong overexpression of AcbB induced only smaller increases of acarbose production compared to medium strong overexpression of AcbB. This may be due to an imbalance in glucose-phosphate-metabolism, occurring upon massive overexpression of AcbB. Overexpression of gtaB might cure this imbalance, and combined overexpression of both, acbB and gtaB therefore plausibly leads to a further increase in acarbose production.
Interestingly, a significant decreased amount of the mass m/z=545 [M-H+] was found in pSETT4tip::gtaB (approx. decrease of 48%), which might correspond to dTDP-4-keto-6-deoxy-D-glucose, the proposed product of AcbB. This may indicate, that the flow through the synthesis strand is more balanced, since the accumulation of this metabolite is reduced in comparison to the empty vector control and AcbB-overexpression mutants (
According to some embodiments, the method according to the first aspect comprises engineering the Actinomycetales strain
According to some embodiments, the method according to the first aspect further comprises engineering the Actinomycetales strain for absent or reduced expression of treY.
According to some embodiments, the method according to the first aspect further comprises
According to some embodiments, the expression cassette according to (iii) and/or (iv) is under the control of a medium strong promoter, as characterized by a normalized glucuronidase activity of between 1x·10−4 and 5×10−5 [L·g−1·min−1] in a glucuronidase assay.
According to a second aspect there is provided an Actinomycetales strain, such as an Actinoplanes strain, for the production of acarbose. According to some embodiments the Actinomycetales strain is a strain generated by a method according to the first aspect. According to some other embodiments the Actinomycetales strain is genetically engineered for absent or reduced expression of extracellular small carbohydrate binding protein Cgt (SEQ ID No. 20).
According to some embodiments the Actinomycetales strain is a Δcgt mutant. A Δcgt mutant is a variant of an Actinomycetales strain wherein the gene Cgt (SEQ ID No. 20) has been at least partially deleted or inverted.
According to some of these or other embodiments the Actinomycetales strain is genetically engineered for absent or reduced expression of at least one gene which is essential for carotenoid synthesis. According to some embodiments the at least one gene which is essential for carotenoid synthesis has been at least partially deleted or inverted. According to some of these embodiments the at least one gene which is essential for carotenoid synthesis comprises at least one gene selected from any of
According to some of these or other embodiments the Actinomycetales strain is genetically engineered for overexpression of MerR-/HTH-transcriptional regulator gene merR (ACSP50_0145, SEQ ID No. 11).
According to some of these or other embodiments the Actinomycetales strain is genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13).
As described elsewhere herein, overexpression of AcbB refers to an increase of AcbB by a factor of at least 1.5 or at least a factor of 2 compared to the control. Preferably, the control is the strain which has not been engineered for the specific overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13). For example, the control does not comprise a vector comprising an expression cassette for AcbB.
For example, the overexpression of the gene product may be an increase during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a factor of a log 2(fold change) of at least 1.5 or at least 2, during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) ≥2 and ≤6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time, such as during the early growth phase and/or during the linear growth phase.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is the increase of the expression of AcbB transcript and/or protein by a log 2(fold change) >6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises a vector for overexpression of AcbB. According to some of these embodiments, the vector is a vector as described herein, preferably according to an aspect described herein.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises an expression cassette for AcbB (SEQ ID No. 13) under the control of a medium strong promoter.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises an expression cassette for AcbB (SEQ ID No. 13) under the control of strong promoter. Preferably, the promoter is not the native promoter of AcbB.
According to some of these or other embodiments the Actinomycetales strain is genetically engineered for overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID No. 19).
Overexpression of GtaB (SEQ ID No. 19) as described elsewhere herein refers to an increase in expression for GtaB compared to the wild type or a specified reference strain/control.
Preferably, the control is the strain which has not been engineered for the specific overexpression of GtaB (SEQ ID No. 19). For example, the control does not comprise a vector comprising an expression cassette for GtaB (SEQ ID No. 19). For example, the overexpression of the gene product may be an increase during the early growth phase and/or during the linear growth phase and/or during the stationary phase, and/or an increase during any other time.
According to some embodiments the overexpression of GtaB is the increase of the expression of GtaB transcript and/or protein by a factor of a log 2(fold change) of at least 1.5, or at least 2, during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of GtaB is the increase of the expression of GtaB transcript and/or protein by a log 2(fold change) ≥2 and ≤6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time, such as during the early growth phase and/or during the linear growth phase.
According to some embodiments the overexpression of GtaB is the increase of the expression of GtaB transcript and/or protein by a log 2(fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the overexpression of GtaB is the increase of the expression of GtaB transcript and/or protein by a log 2(fold change) >6 during the early growth phase, during the linear growth phase, during the stationary phase or an increase during any other time.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of GtaB comprises a vector for overexpression of GtaB. According to some of these embodiments, the vector is a vector as described herein, preferably according to an aspect described herein.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of GtaB (SEQ ID No. 19) comprises an expression cassette for GtaB (SEQ ID No. 19) under the control of a medium strong promoter.
According to some embodiments the Actinomycetales strain genetically engineered for overexpression of GtaB (SEQ ID No. 19) comprises an expression cassette for GtaB (SEQ ID No. 19) under the control of strong promoter. Preferably, the promoter is not the native promoter of GtaB.
According to a third aspect there is provided an Actinomycetales strain, such as an Actinoplanes strain, for the production of acarbose for use in the production of acarbose.
According to some embodiments there is provided a method for the production of acarbose, wherein the method comprises the use of an Actinomycetales strain according to the second aspect.
For genetic engineering of Actinoplanes, an expression system is required for the overexpression of singular or multiple genes. According to a fourth aspect there is provided an expression vector for Actinoplanes.
According to some embodiments, the vector according to the fourth aspect comprises a medium strong promoter characterized by a normalized glucuronidase activity of at least 1x·10−4 [L·g-1·min-1] in a glucuronidase assay. In some embodiments, the medium strong promoter is selected from efp according to SEQ ID No. 92, cdaR according to SEQ ID No. 97, rpsL according to SEQ ID No. 99, rpsJ according to SEQ ID No. 93, cgt according to SEQ ID No. 91, or tipA according to SEQ ID No. 81.
According to some embodiments, the vector according to the fourth aspect comprises a strong promoter characterized by a normalized glucuronidase activity of at least 5x·10−4 [L·g-1-min-1] in a glucuronidase assay. In some embodiments, the strong promoter is selected from apm according to SEQ ID No. 96, ermE* according to SEQ ID No. 98, katE according to SEQ ID No. 94, moeE5 according to SEQ ID No. 95 or gapDH according to SEQ ID No. 82.
To find further suitable promoters, that allow medium to strong gene expression, a promoter screening can be carried out by use of the screening system of Horbal et al. (2013) and Myronovskyi et al. (2011), which is based on the reporter GusA cloned in a pSET152-vector system, cf.
In some embodiments the vector according to the first aspect comprises an expression cassette.
Preferably the vector comprises an expression cassette for AcbB (SEQ ID No. 13) and/or an expression cassette for GtaB (SEQ ID No. 19) and/or an expression cassette for MerR.
In some embodiments, the expression cassette may furthermore comprise a lacZα-gene under control of the lac-promoter. The lacZα-gene encodes a catalytic domain of a β-galactosidase, that enables quick selection of the integration of a target sequence by blue/white-selection in the cloning strain Escherichia coli DH5αMCR (NC_017638.1) (Grant et al. 1990).
Without being bound by theory, the vector according to the current aspect comprises elements for vector replication, transfer, maintenance and selection. In some embodiments, at least one of these elements is derived from pSET152.
In some embodiments, the vector according to the current aspect comprises parts of the sequence of the pSET152 vector of Bierman et al. (1992).
Preferably, the vector does not comprise putative antisense promoters according to SEQ ID NO 108 and/or SEQ ID No. 109. These antisense promoters were identified by the inventors by sequencing of a 5′-primary transcript library and impair suitability of the vector pSET152. In brief, identification occurred by sequencing of an enriched primary transcript library. The two putative promoters were identified behind the gene of interest in antisense orientation (
Furthermore, a T4-terminator was introduced behind the expression cassette in opposite orientation to prevent further putative antisense reads (cf. e.g.
In some embodiments the vector comprises the φC31 integrase gene int. In some of these embodiments the φC31 integrase gene int is derived from pSET152. In some embodiments, the vector according to the first aspect furthermore comprises the attachment site attP. The integrase of the φC31 integrase gene int mediates the integration of the vector into the host chromosome at a distinct genomic location by catalyzing the targeted and unidirectional recombination of two attachment sites: attP, localized on the vector, and attB, localized in the host chromosome in the gene ACSP50_6589 (former: ACPL_6602) (te Poele et al. 2008; Gren et al. 2016). Without being bound by theory, after integration, the vector is flanked by the attachment site left (attL) and right (attR), which are derived from attP-attB-recombination (te Poele, Bolhuis und Dijkhuizen 2008).
In some embodiments the vector comprises an origin of transfer such as the origin of transfer (incP) and/or a relaxosome gene such as the relaxosome gene traJ. In some of these embodiments the origin of transfer such as the origin of transfer (incP) and/or the relaxosome gene, such as traJ are derived from pSET152. The origin of transfer and the relaxosome gene enable the transfer of the plasmid from the donor strain (e.g. Escherichia coli ET12567/pUZ8002 (Kieser et al. 2000)).
In some embodiments the vector according to the first aspect comprises an origin of replication such as the high-copy-number ColE1/pMB1/pBR322/pUC origin of replication (ori). In some of these embodiments the origin of replication such as the high-copy-number ColE1/pMB1/pBR322/pUC origin of replication (ori) is derived from pSET152. The origin of replication such as the high-copy-number ColE1/pMB1/pBR322/pUC origin of replication (ori) enables replication of the plasmid in the cloning strain (Escherichia coli DH5αMCR) and donor strain (Escherichia coli ET12567/pUZ8002).
In some embodiments the vector according to the first aspect comprises at least one resistance marker such as a resistance marker mediating apramycin resistance (aac(3)IV, apmR).
Resistance markers mediating apramycin resistance (aac(3)IV, apmR) can be used for selection.
According to some embodiments according to the fourth aspect the expression vector comprises at least one element of pSET152, such as (a) the φC31 integrase gene int according to SEQ ID No. 85, (b) the origin of transfer (incP) according to SEQ ID No. 87, (c) the relaxosome gene traJ according to SEQ ID No. 88, or (d) the high-copy-number ColE1/pMB1/pBR322/pUC according to SEQ ID No. 89, and furthermore does not comprise putative antisense promoters according to SEQ ID NO 108 and SEQ ID No. 109.
According to some embodiments according to the fourth aspect the expression vector comprises (a) the φC31 integrase gene int according to SEQ ID No. 85, and (b) the origin of transfer (incP) according to SEQ ID No. 87, and (c) the relaxosome gene traJ according to SEQ ID No. 88, and (d) an origin of replication, such as the high-copy-number ColE1/pMB1/pBR322/pUC, origin of replication (ori) according to SEQ ID No. 89 and (e) optionally at least one resistance marker, such as a resistance marker mediating apramycin resistance, such as aac(3)IV according to SEQ ID No. 90, apmR, and (f) optionally at least one T4-terminator, and (g) optionally, wherein the vector does not comprise putative antisense promoters according to SEQ ID NO 108 and/or SEQ ID No. 109.
According to some embodiments, the vector comprises the sequence according to SEQ ID No. 110 or SEQ ID No. 111. According to some embodiments, the vector comprises the sequence according to SEQ ID No. 110 or SEQ ID No. 111, or a fragment thereof.
In some embodiments, the vector is excelled by an easy cloning mechanism allowing integration of different promoters. By this, the system can be quickly adapted to further species, e.g. production strains of Acarbose.
General Tools and Methods
Strains and Plasmids
All strains used in this work are listed in Table E1. Recombinant strains used or created in this work are listed in Table E2, Table E3 and Table E4 (plasmid-based expression systems in Table E2, deletion and integration constructs cloned and stored in E. coli DH5αMCR in Table E3, deletion and integration mutants of Actinoplanes sp. SE50/110 in Table E4).
Escherichia coli DH5αMCR
Escherichia coli
E. coli
E. coli
Media and Cultivation Conditions
Unless otherwise specified, all chemicals and media components were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany), Sigma-Aldrich (St. Louis, USA), SERVA Electrophoresis GmbH (Heidelberg, Germany) or VWR International (Pennsylvania, USA).
Preparation of Glycerol Stocks of Actinoplanes sp. SE50/110
For preparation of glycerol stocks, Actinoplanes sp. SE50/110 (ATCC 31044) was grown in the complex medium NBS (11 g·L−1 glucose·1H2O, 4 g·L−1 peptone, 4 g·L−1 yeast extract, 1 g·L−1 MgSO4·7H2O, 2 g·L−1 KH2PO4, 4 g·L−1 K2HPO4) and mixed 2:3 with sterile 86% (v/v) glycerol. Glycerol stocks are stored at −80° C.
Growth on Solid Media and Preparation of Spore Solutions
For spore formation, 200-300 μL of a glycerol stock were grown on agar plates of soy flour medium (SFM-agar) (20 g·L−1 soy flour (SOBO® Naturkost (Cologne, Germany)), 20 g·L−1 D-mannitol, 20 g·L−1 Bacto™ agar (Becton-Dickinson, Heidelberg, Germany), 167 μL 10 N NaOH in tap water). Spores could be harvested after 5-7 days of incubation at 28° C. by washing them off in 3 mL ddH2O with a cotton swab, like described by Wolf et al. (2016).
Preparation of Minimal Medium
Maltose minimal medium (72.06 g·L−1 maltose 1H2O, 5 g·L−1 (NH4)2SO4, 0.184 g·L−1 FeCl2·4H2O, 5.7 g·L−1 Na3C6H5O7·2H2O, 1 g·L−1 MgCl2·6H2O, 2 g·L−1 CaCl2·2H2O, trace elements (final concentration: 1 μM CuCl2, 50 μM ZnCl2, 7.5 μM MnCl2 dissolved in 1 M HCl) and phosphate buffer consisting of 5 g·L−1 each K2HPO4 and KH2PO4 in ddH2O) was prepared and filter sterilized following the protocol of Wendler et al. (2013).
For substitution of the carbon source maltose, 79.2 g·L−1 glucose·1H2O, 72.0 g·L−1 C-pur (Cerestar 01908, CerestarGmbH, Krefeld, Germany), 71.9 g·L−1 galactose, 68.4 g·L−1 cellobiose, 71.9 g·L−1 D-arabinose or 72.0 g·L−1 D-lactose were used respectively, instead of maltose-monohydrate. Mixtures of maltose and glucose were prepared in the ratio of 90:10, 80:20 and 50:50 (v/v).
For the starch medium, a 4% (w/v) opalescent solution of “starch soluble” from Acros Organics (part of Thermo Fisher Scientific, Geel, Belgium) was generated. For this, sterile water was preheated to 90° C. in a water bath and the weighed portion of starch added with stirring.
Afterwards, the residual media components were added. To allow comparison to the starch-cultivation, a maltose minimal medium was created with comparable C-molarity (here net weight of 44.4 g·L−1 maltose 1H2O). Media of different pH and osmolarity were created by addition of correcting agents (HCl or NaOH), by varying of the concentration of the carbon-sources maltose respectively by addition of inositol, which is not metabolized according to our study (data not shown).
Furthermore, minimal media with 1 g·L−1, 2 g·L−1, 3 g·L−1, 4 g·L−1 and 5 g·L−1 “starch soluble” from Acros Organics were created for cultivation under limited carbon-source.
The pH and osmolarity of all media were determined by the pH-meter Calimatic of Knick GmbH (Berlin, Germany) and the Osmomat 3000 of Gonotec GmbH (Berlin, Germany) according to the manufacturer's instructions.
Shake Flask Cultivation
Cultivations were performed in 250 mL Corning® Erlenmeyer baffled cell culture flasks at 28° C. and 140 rpm for seven days in the GFL shake-imcubators 3032 or 3033 (Burgwedel, Germany).
For inoculation of 50 mL medium, 1 mL spore solution of an OD=3-5 was used. Cell dry weights were determined like described by Wolf et al. (2017a). The supernatant was stored at −20° C. for later analysis.
Miniaturized cultivation in the BioLector system of m2p-labs GmbH (Baesweiler, Germany)
Comparative growth experiments were performed in a 1 mL reaction volume in a 48-well FlowerPlate covered by a gas-permeable sealing foil (m2p-labs GmbH, Baesweiler, Germany) and incubated for 1 week at 28° C. and 800 rpm in the RoboLector® of m2p-labs. Growth was recorded by the backscatter signal. For determination of final cell dry weights, 800 μL of each well was sampled in a weighed reaction tube (14,000 g, 2 min), washed with deionized water and dried for 1 day at 60-70° C. The supernatant was stored at −20° C. for later analyses.
Recombinant DNA Work
Unless otherwise specified, plasmid construction and assembly was performed by Gibson Assembly (Gibson et al. 2009). Fragments were amplified by PCR (Phusion® High-Fidelity PCR Master Mix with GC Buffer, NEB, Ipswich, Mass., USA) in the Eppendorf thermocycler vapo.protect (Hamburg, Germany) and treated with DpnI (Thermo Fisher Scientific, Waltham, Mass., USA), when necessary. Purification of PCR products and gel extracts was performed by use of the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, DQren, Germany). Equimolar amounts of the DNA fragments were added to the Gibson Assembly Master Mix in a ratio of 1:4.
The master mix consists of 0.64 μL T5 Exonuclease (10 U·μL−1, NEB, Ipswich, Mass., USA), 20 μL Phusion High-Fidelity DNA Polymerase (2 U·μL−1, Thermo Fisher Scientific, US), 160 μL Taq DNA Ligase (NEB, Ipswich, Mass., USA), 699.36 μL aqua distilled and 320 μL isothermal reaction buffer (25% PEG-8000, 1 mL 1 M Tris-HCl, 100 μL 1 M MgCl2, 100 μL 1 M DTT, 20 μL each 1 mM dNTP, 200 μL NAD). The sample was incubated at 50° C. for at least 1 h and subsequently transferred to Escherichia coli DH5αMCR by chemical transformation according to a protocol of Beyer et al (2015). Selection of E. coli was performed on Luria/Miller broth medium with 15 g·L−1 agar-agar (Carl Roth, GmbH&Co.KG, Karlsruhe, Germany)) and 50 mg·L−1 apramycin-sulfate.
Positive colonies were tested by PCR and gel-electrophoresis as well as by Sanger sequencing by our in-house sequencing core facility.
Construction of Plasmids for the gusA Reporter System
For the construction of plasmids for the gusA reporter system see Schaffert et al. (2019).
Construction of the Novel pSETT4 Expression System
For cloning of the novel pSETT4 expression system, the pSET152 vector of Bierman et al. (1992) was used as template. The vector backbone was linearized by PCR (Table E5).
The cloning cassette, consisting of the gapDH-promoter, a lacZ-gene under control of the lac-promoter and several restriction sites flanked by three T4-terminators, was ordered as string DNA at Integrated DNA Technologies (Iowa, USA). Due to the complex structure, the cassette was ordered in three parts and assembled by GeneSOEing (Horton 1995) by use of the primers in Table E5. Finally, backbone and insert were assembled by Gibson Assembly (Gibson et al. 2009). The novel vector system was named pSETT4gap.
For exchange of the gapDH-promoter by the tipA-promoter, pSETT4gap was digested with NdeI and KpnI and treated with shrimp alkaline phosphatase following the instructions of the supplier.
All enzymes were purchased from Thermo Fisher Scientific (Waltham, Mass., USA). The tipA-promoter was amplified from pSETGUS (Myronovskyi et al. 2011) by use of the primers tipA_GAF and tipA_GAR and assembled with the linearized backbone by Gibson assembly (Gibson et al. 2009). The vector was named pSETT4tip (cf.
Overexpression of Single Genes in the Novel pSETT4 Expression System
For the overexpression of single genes, the insert was amplified by PCR (Table E6). The vector (pSETT4gap or pSETT4tip), was digested with BsaI (NEB, Ipswich, Mass., USA) and assembled with the insert by Gibson Assembly (Gibson et al. 2009). For expression of the acbB gene under control of the native promoter, the vector backbone pSETT4gap was digested with BsaI and NdeI, leading to the linearization of the vector under removal of the promoter. The gene of interest and the native promoter were amplified by use of the primers in Table E6 and assembled with the vector backbone by Gibson Assembly (Gibson et al. 2009).
Construction of pCRISPomyces-2 Deletion and Integration Vectors
For the construction of deletion and integration mutants by CRISPR/Cas9 technique, the plasmid pCRISPomyces-2 (Cobb et al. 2015) was used according to a protocol of Wolf et al. (2016). The spacer and its reverse complement were ordered at metabion GmbH (Steinkirchen, Germany) or Sigma-Aldrich (Taufkirchen, Germany) as oligonucleotides with overlap (Table E7).
The oligonucleotides were annealed to a double-strand and assembled with the plasmid by Golden Gate Assembly (Engler et al. 2008) according to the protocol of Cobb et al. (2015). For repair of the Cas9-induced double-strand break, a DNA template was cloned into the vector backbone by Gibson Assembly (Gibson et al. 2009). As DNA template, flanking sequences up- and downstream of the target gene (each round about 1 kB) were amplified by PCR (Table E8) from genomic DNA.
Deletion of the gene cgt by CRISPR/Cas9 technique
For the construction of a Δcgt (ΔACSP50_5024) deletion mutant by CRISPR/Cas9 technique (clustered regular interspaced short palindromic repeats/CRISPR-associated endonuclease 9), the plasmid pCRISPomyces-2 was used (Cobb et al. 2015). The spacer sequence was selected according to Wolf et al. (2016) and ordered as oligonucleotides together with its reverse complement at metabion GmbH (Steinkirchen, Germany) (spacer_1: 5′-acgcAGCGTCGCCCGCTGGGAGAA-3′, spacer_2: 5′-aaacTTCTCCCAGCGGGCGACGCT-3′).
The oligonucleotides were annealed to a double-strand and assembled with the plasmid by Golden Gate Assembly (Engler et al. 2008) by use of BsaI (NEB, lpswich, MA, USA) according to the protocol of Cobb et al. (2015) (Cobb et al. 2015). For repair of the Cas9-induced double-strand break, a deoxyribonucleic acid (DNA) template was cloned into the XbaI-linearized vector by Gibson Assembly (Gibson et al. 2009). As DNA template, flanking sequences up- and downstream of the target gene (each round about 1 kB) were amplified by polymerase chain reaction (PCR) with the Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB, Ipswich, Mass., USA) (Primer sequences: cgt_flank1_fw: 5′-tcggttgccgccgggcgttttttatCCGGTACCCTGCTCCTCGTC-3′, cgt_flank1_rv: 5′-gtgacgcattgacgcaggtcGAGGGATATGGCTCAGATAC-3′, cgt_flank2_fw: 5′-gtatctgagccatatccctcGACCTGCGTCAATGCGTCAC-3′, cgt_flank2_rv: 5′-gcggcctttttacggttcctggcctACCTGACCCTGCTGAAATGG-3′). For Gibson Assembly, the DNA fragments (flank_1: 1101 bp and flank_2: 982 bp) were mixed equimolar added in a ratio of 1:4 to the Gibson Assembly Master Mix consisting of 0.64 μL T5 Exonuclease (10 U/μL, NEB, Ipswich, Mass., USA), 20 μL Phusion High-Fidelity DNA Polymerase (2 U/μL, Thermo Fisher Scientific, US) and 160 μL Taq DNA Ligase (40 U/μL NEB, Ipswich, Mass., USA), 699.36 μL aqua distilled and 320 μL isothermal reaction buffer (25% PEG-8000, 1 mL 1 M Tris-HCl, 100 μL 1 M MgCl2, 100 μL 1 M DTT, 20 μL each 1 mM dNTP, 200 μL NAD). After incubation at 50° C. for at least 1 h, the reaction mix was transferred to Escherichia coli DH5αMCR by chemical transformation according to a protocol of (Beyer et al. 2015). Growth and selection of E. coli was performed by plating them on Luria/Miller broth (LB-media) with 15 g·L−1 agar-agar Kobel (both: Carl Roth, GmbH&Co.KG, Karlsruhe, Germany)) supplemented with 50 mg·L−1 apramycin-sulfate. Plates were incubated for 10-14 h at 37° C. Apramycin-resistant colonies were tested by PCR and gel-electrophoresis first, and second by Sanger sequencing by our in-house sequencing core facility (primer sequences for PCR: for: 5′-GGCGTTCCTGCAATTCTTAG-3′, rev: 5′-TCGCCACCTCTGACTTGAGC-3′, walking primer for sequencing: w1: 5′-CGCTGATCTTCAGCTTCC-3′, w2: 5′-GCCTTCACCTTCCATCTG-3′, w3: 5′-TCGGGAAAGCCGCCGGAG-3′)).
Conjugal transfer to Actinoplanes sp. SE50/110
Competent Actinoplanes sp. SE50/110 cells were prepared from a freshly grown NBS-culture (see above). Cells were washed twice in 10% (w/v) ice-cold sucrose and twice in ice-cold 15% (v/v) glycerol. Finally, the cells were taken up in 15% (v/v) ice-cold glycerol (by addition of round about the four-fold volume of the cell pellet), aliquoted to 100 μL in reaction tubes and snap-frozen in liquid nitrogen. The competent Actinoplanes cells are stored at −80° C.
For conjugation, Escherichia coli ET12567/pUZ8002 (Kieser et al. 2000) was used. After transfer of the desired construct into E. coli ET12567/pUZ8002 according to Beyer et al. (2015) and selection on LB agar plates supplemented with 50 mg·L−1 apramycin-sulfate, 50 mg·L−1 kanamycin-sulfate and 15 mg L−1 chloramphenicol, cells were grown in liquid culture (LB-medium with the same supplements) and harvested at an optical density of 0.4-0.6. The cells were washed twice in ice-cold LB medium and mixed with competent cells of Actinoplanes sp. SE50/110. The cell suspension was plated on SFM agar plates. After 20-24 h of incubation at 28° C., 1 mL 500 mg·L−1 apramycin-sulfate dissolved in ddH2O was distributed on the plate with a sterile swab. First exconjugants of Actinoplanes sp. SE50/110 can be observed after 1 week. Exconjugants were transferred to an SFM agar plate supplemented with 50 mg·L−1 apramycin-sulfate. Repeated streaking is performed for several times to purify Actinoplanes exconjugants from E. coli. To expedite this process, 50 mg·L−1 fosfomycin or trimethoprim can be supplemented to the medium to get rid of the donor strain.
Plasmid Curing to Obtain Marker-Free CRISPR/Cas9 Deletion/Integration Mutants of Actinoplanes sp. SE50/110
Plasmid curing was performed according to the protocol of Wolf et al. (2016) by cultivation in the complex medium NBS at elevated temperatures. Colonies were tested for the presence of the plasmid by parallel streaking on apramycin-containing and apramycin-free SFM plates. Apramycin-sensitive exconjugants were tested for the deletion by PCR (primer sequence data not shown). The PCR fragment was excised from the gel and sequenced by our in-house Sanger sequencing core facility.
Additionally, also genomic DNA of the deletion or integration mutant was sequenced by the Oxford Nanopore technique (Oxford, UK) to exclude off-target effects. For this, genomic DNA of an NBS-grown culture was isolated with the NucleoSpin® Microbial DNA kit (Macherey-Nagel, DQren, Germany). A library was prepared with help of the 1D Genomic DNA by ligation-kit (Oxford Nanopore, Oxford, UK).
Deletion System Based on Homologous Recombination and Counterselection with the Cytosine Deaminase CodA.
Vector integration into genes of the acb gene cluster has occurred by use of the replicative vector pKC1139. Based on this observation, a novel deletion system using homologous recombination was developed and tested by example of the gene cgt (ACSP50_5024).
A vector backbone with origin of transfer (ncP) and relaxosome gene traJ was used to allow conjugation into Actinoplanes sp. SE50/110. In this work, two different antibiotic resistance markers mediating apramycin and kanamycin resistance were tested for selection: aph(3′)II (Kan®, kanamycin) and aac(3)IV (Apm®, apramycin). Furthermore, the high-copy-number ColE1/pMB1/pBR322/pUC origin of replication was integrated to allow replication in the donor strain E. coli. The ori, the oriTncP, tra gene and resistance cassettes were taken from pRT802 respectively pRT801 (Gregory et al. 2003). Since no replicon for replication in Actinoplanes sp. SE50/110 neither an integrase gene with attachment site are contained in the novel deletion system, the vector can only be maintained in Actinoplanes sp. SE50/110 when being integrated into the genome by homologous recombination (
The novel deletion system was successfully tested for the gene cgt, which was shown by colony PCR and ONT-sequencing. The proportion of deletion mutants after successful second crossover was between 25% and 32%. The workflow is illustrated in
Analytical Methods
Acarbose Quantification from the Supernatant by High Performance Liquid Chromatography (HPLC)
Supernatants of maltose-grown cultures of Actinoplanes ssp. were centrifuged (20,000 g, 2 min), mixed 1:5 with methanol by vortexing and centrifuged again to remove the precipitate (20,000 g, 2 min). The samples were transferred to HPLC vials and analyzed in the HPLC system 1100 series of Agilent (G1312A Binary Pump Serial #DE43616357, G1329A ALS autosampler Serial #DE43613/10, G1315A diode-array detector (DAD) Serial #DE72002469). As stationary phase the Hypersil APS-2 column (125×4 mm, 3 μm particle size) of Thermo Fisher Scientific Inc. (Waltham, Mass., USA) was used, heated to 40° C. As mobile phase an isocratic flow of 1 mL·min−1 68% acetonitrile (solvent B) and 32% phosphate buffer (0.62 g·L−1 KH2PO4 and 0.38 g·L−1 Na2HPO4·2H2O) (solvent A) was applied. 40 μL of each sample was injected and separated in a 10 min run. Detection of acarbose was carried out with a DAD detector at 210 nm (Reference 360 nm) and quantified from the peak areas of a calibration curve.
Liquid Chromatography-Mass Spectrometry (LC-MS)
Sample Preparation for Analysis of Intracellular Metabolites
Triplicates of Actinoplanes sp. SE50/110 strains were grown in maltose minimal medium for at least 4 days. 10 mL of the culture were quickly filtrated through filtering paper by a Buchner funnel and washed with 2.63 g·L−1 NaCl solution. Cells were transferred into pre-weighted round bottom screw-cap tubes, snap-frozen in liquid nitrogen and stored at −80° C. Cells were dried overnight in the Centrifugal Evaporator (SpeedVac) of Thermo Fisher Scientific (Waltham, Mass., USA). 4 mg dried cells were transferred into a fresh 2 mL screw-cap tube containing round about 500 μL of a mixture of zirconia/silica micro beads of the sizes 0.1 mm, 0.05 mm and 0.01 mm (Bio Spec Products Inc., Bartlesville, USA). 700 μL 80% MeOH was added to the cells and beads. Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, Mass., USA) for three times 30 s at speed setting 6.5. Samples were cooled for 5 min on ice in between. The cell suspension was centrifuged for 5 min at 13,000 g and 4° C. 500 μL of the supernatant was transferred into HPLC vials, dried under nitrogen flow and taken up in 50 μL distilled water.
Sample Preparation for Analysis of Extracellular Acarviosyl-Metabolites
The sample preparation was conducted according to a protocol described by Ortseifen (2016). Sugars and pseudo-sugars were enriched from 10 mL of the supernatant by solid phase extraction using the Chromabond® Easy columns (Macherey-Nagel, DQren, Germany, REF 730753). The columns were equilibrated with 3 mL methanol, afterwards washed with 3 mL distilled water before loading of the sample. Unspecific bound metabolites were rinsed by 3 mL 95% (v/v) methanol. Elution was conducted in 3 mL methanol.
LC-ESI-MS of Intracellular and Extracellular Metabolites
For LC-MS, the LaChromUltra (Hitachi Europe Ltd., UK) HPLC system coupled to a microTOF-Q hybrid quadrupole/time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) was used, which was equipped with an electrospray ionization (ESI) source.
For the analysis of intracellular metabolites, 2 μL of the sample was separated with the SeQuant® ZIC®-pHILIC 5 μm Polymeric column (150×2.1 mm) (Merck, Darmstadt, Germany). Eluent A (20 mM NH4HCO3, pH 9.3, adjusted with aqueous ammonia solution) and eluent B (acetonitrile) were applied at a flow rate of 0.2 mL·min−1 by use of the following gradient: 0 min B: 90%, 30 min B: 25%, 37.5 min B: 25%, 40.0 min B: 80%.
As standards for the peak identification, 2 μL of 10 μM of UDP-glucose, glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate and dTDP-glucose were injected.
For the analysis of extracellular acarviosyl-metabolites, 10 μL of the sample was separated with the Cogent Diamond Hydride™ HPLC column (MicroSolv Technology Corporation; 150 mm×2.1 mm; 3 μL particle size). Eluent A (50% (v/v) acetonitrile, 50% (v/v) H2O und 0.1% (v/v) formic acid) and eluent B (90% (v/v) acetonitrile, 10% (v/v) H2O und 0.1% (v/v) formic acid) were applied at a flow rate of 0.4 mL·min−1 by use of the following gradient: 0 min B: 100%, 8 min B: 0%, 13 min B: 0%, 15.5 min B: 100%, 18 min B: 100%.
The ESI source was operated in the negative ionization mode for analysis of intracellular metabolites and in the positive ionization mode for analysis of extracellular acarviosyl-metabolites. The temperature of the dry gas and the capillary was set to 180° C. The scan range of the MS was set to 200-1,000 m/z (intracellular metabolites) respectively 50-3,000 m/z (extracellular acarviosyl-metabolites)
The peak areas of specific masses were integrated by use of the software Compass™ (Bruker Daltonics, Bremen, Germany). Peaks were normalized on the weighed amount of dried cells (intracellular metabolites) respectively the cell dry weight at sampling time (extracellular acarviosyl-metabolites).
Extraction and Analysis of Carotenoids
Extraction
Cell pellets from Actinoplanes sp. SE50/110 were transferred into a 2 mL screw-cap tube with round about 500 μL of a mixture of zirconia/silica micro beads of the sizes 0.1 mm, 0.05 mm and 0.01 mm (Bio Spec Products Inc., Bartlesville, USA). 1 mL acetone or methanol was added as extracting solvent. Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, Mass., USA) for three times 45 s at speed setting 6.5. Samples were cooled for 5 min on ice in between. The homogenized cell suspension was centrifuged for 20 min at 13,000 g and 4° C. The supernatants were transferred into glass vials. For HPLC-analysis, mixtures of the acetone- and methanol-extracts were created in the ratio of 7:3 and transferred into a novel glass vial.
Thin Layer Chromatography (TLC) and Spectral Analysis
50 μL of the extracted carotenoids were applied in 5 μL-steps onto a silica gel matrix (HPTLC-HL, Cat. 58077, Analtech Inc., Newark, USA) and incubated in a TLC-chamber filled with 100 mL petroleum, 11 mL isopropanol and 50 μL water. The run was carried out in darkness. After drying of the TLC-plate, bands were stripped off with a scalpel and transferred into a novel tube. After addition of 1 mL ethanol, the absorption spectrum was analyzed by use of the Genesys 10S UV-Vis spectrophotometer of Thermo Fisher Scientific (Waltham, Mass., USA).
HPLC Analysis of Carotenoids with Absorbance Scan
Carotenoids were separated by reversed-phase HPLC according to Henke et al. (2017) and Heider et al. (2014) using the Agilent 1200 series HPLC system (Agilent Technologies GmbH&Co. KG, Böblingen, Germany) including diode array detector (DAD) for the UV-Vis spectrum. 20 μL sample volume was applied to a flow of 0.5 mL−min−1. As stationary phase a pre-column (10×4 mm MultoHigh 100 RP18-5) and a main column (ProntoSIL 200-5 C30, 250×4 mm) from CS ChromatographieService GmbH (Langerwehe, Germany) were used, like described before (Heider et al. 2014; Henke et al. 2017).
Following gradient was applied: 0 min A: 100%, 32 min A: 75%, 47 min A: 0%, 70 min A: 0%, 75 min A: 100%, with eluent A consisting of 0.1 M ammonium acetate in deionized water and methanol in the ratio of 15:85 (v/v). Eluent B consists of a mixture of methanol, acetonitrile and acetone in the ratio of 44:43:13 (v/v). Detection of carotenoids was conducted at 470 nm. Additionally, wavelength scans between 360 nm and 700 nm were performed each second during the run.
Assays
Promoter Screening Experiment by Spectrophotometric Measurement of the Glucuronidase Activity
Two different types of glucuronidase assay were carried out: one with protein raw extract and one with entire cells. The protocols described by Horbal et al. (2013) and Siegl et al. (2013) were adapted to Actinoplanes sp. SE50/110. The substrate 5-bromo-4-chloro-3-indolyl-p-D-glucuronide (X-Gluc, AppliChem GmbH, Darmstadt, Germany) was chosen, as the substrate p-nitrophenyl-D-glucuronide turned out to dissociate under our assay conditions.
Growth Conditions and Sample Preparation
Actinoplanes mutants carrying promoter constructs with gusA gene, were cultivated for one week in maltose minimal medium, like described above. The assays were conducted during growth phase. 500 μL of each culture was sampled for an assay with entire cells. 1 mL was sampled for an assay with protein raw extract and transferred to a screw cap tube containing zirconia/silica micro beads (Bio Spec Products Inc., Bartlesville, USA) of the sizes 0.1 mm and 0.05 mm. Cells were disrupted in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, Mass., USA) for two times 30 s at speed setting 6.5 and 5 min on ice in between. After centrifugation, the lysate was transferred to a new reaction tube and centrifuged. The supernatant was used for a cell-free assay. Total protein quantification was carried out by a Bradford assay (see above).
Glucuronidase (Gus) Assay
The gus assay was performed in a black microtiter plate (96 well PS F-bottom pCLEAR, black, med. binding, Greiner Bio-One, Kremsmünster, Österreich, REF 655096). 100 μL of each sample (either cell suspension or lysate) was pipetted in three wells, of which one serves as negative control and two as technical replicates. The gus buffer (50 mM phosphate buffer pH 7.0 (5.136 g·L−1 Na2HPO4·2H2O, 3.299 g·L−1 NaH2PO4·2H2O) with 5 mM DTT and 0.1% Triton-X-100) was complemented with 2 mM substrate X-Gluc (stock solution: 0.2 M in DMF). 100 μL was added to 100 μL of the sample. For the negative control, 100 μL gus buffer without substrate was added. Beside of the individual negative control of each sample, also medium and substrate controls were prepared.
The microtiter plate was measured in a pre-warmed Tecan reader Infinite M200 (Ref 30016056, Tecan Group AG, Männedorf, Switzerland) (37° C.) for 3 hours (assay with entire cells), respectively for 2 hours (assay with lysate). The absorption maxima of indigo were measured at 610 and 660 nm. After discounting the absorption value of all controls, the slope of each absorption curve was calculated by linear regression and normalized either on cell dry weight (assay with entire cells) or on whole protein amount (assay with lysate). The normalized slope was used to compare the p-glucuronidase activities in the different mutants.
Screening experiments in the Biolog® OmniLog Phenotypic Microarray System
Pre-screening experiments were performed in the Biolog® OmniLog Identification System (Hayward, Calif., USA) to evaluate respiration on different carbon sources (panel PM1 and PM2). Actinoplanes sp. SE50/110 wild type and the deletion mutant Δcgt were grown on SFM agar plates, as described elsewhere herein. Cells were harvested by use of a sterile swab and diluted in the inoculating fluid IF-0a for PM1 and PM2. The turbidity of the cell suspension was checked to achieve 80% transmittance in the turbidimeter of Biolog®, according to manufacturer's protocol. 2.32 mL of the cell suspension was added to 20 mL IF-0a, 0.24 mL 0.5 M MgCl2, 0.24 mL 0.5 M Na2SO4, 0.24 mL 1.5 M NH4Cl, 0.24 mL 1.0 M Na3PO4, 0.24 mL aqua distilled, 0.24 mL Biolog redox dye mix G, and 0.24 mL metal ion cocktail (5.0 mM each: ZnCl2·7H2O, FeCl2·6H2O, MnCl2·4H2O, CaCl2·2H2O), according to the manufacturer's protocol. The PM panels were inoculated with 100 μL per well of the prepared solution and incubated for 1 week in the OmniLog system (Mode 71000 Serial #406) at 28-30° C. Data evaluation was carried out with the manufacturer's software (Kinetic Analysis, Biolog and Omnilog 2.3, Biolog).
RNA Work
Sampling and RNA Isolation
For transcriptome analysis, 2×1 mL culture were taken during growth phase, separated from the supernatant by centrifugation (10 s) and snap-frozen in liquid nitrogen. Pellets were stored at −80° C. until further processing.
For isolation of ribonucleic acid (RNA), frozen cell pellets were resuspended in 500 μL LB-buffer (NucleoSpin® RNA Plus, Macherey-Nagel, DQren, Germany) and transferred to 2 mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, Calif., USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, Mass., USA) for three times 20 s at speed setting 6.5 and 5 min on ice in between.
Subsequently, the cell suspension was centrifuged for 5 min at 13,000 g and 4° C. The supernatant was used for RNA extraction using the NucleoSpin® RNA Plus kit in combination with rDNase Set (Macherey-Nagel, DQren, Germany) for an on-column DNA digestion. After clean-up and elution according to the manufacturer's protocol, the DNA-digestion was repeated (in solution) and the sample cleaned up again by use of the same kit. With two primer pairs binding to the genomic DNA of Actinoplanes sp. SE50/110 and amplifying small fragments at round about 200-300 nt, the sample was tested for residual DNA. DNA digestion and RNA clean-up was repeated, if necessary. The quantity of RNA was analyzed with the NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany).
Reverse Transcription Quantitative PCR
Reverse transcription quantitative PCR was carried out according to the protocol of Wolf et al. (2017a) by use of SensiFast SYBR No-Rox One-Step kit (Bioline, London, UK) and 96 well lightcycler plates (Sarstedt, Numbrecht, Germany) in a LightCycler 96 System of Roche (Mannheim, Germany). The relative RNA amount was normalized on total RNA (100 ng) and calculated as 2−ΔCq. ΔCq is the difference of the mean Cq in the mutant strain compared to the control strain. The primers in Table E9 were used for determination of the relative transcription of a gene.
Whole Genome Oligonucleotide Microarray
Whole genome oligonucleotide microarrays were performed according to a protocol of Wolf et al. (2017a), who adapted the hybridization procedure to the high G+C content of Actinoplanes sp. SE50/110.
RNA of triplicates was isolated and equimolar pooled (total amount of 5 μg pooled RNA in 12 μL). For cDNA synthesis, labeling and microarray hybridization the Two-Color Microarray-Based Prokaryote Analysis FairPlay III Labeling kit (Version 1.4, Agilent Technologies, Santa Clara, Calif., USA) was used according to the manufacturer's instructions with practical adjustments described by Wolf et al. (2017a). The Amersham CyDye mono-reactive dye packs (GE Healthcare, Little Chalfont, UK) were utilized for labeling. A custom whole genome oligonucleotide microarray representing the coding sequence of Actinoplanes sp. SE50/110 was used, which was designed by Wolf et al. (2017a) (4×44 K format, 43,803 features representing 8,238 genes and 1,417 control spots, supplier: Agilent Technologies, Santa Clara, Calif., USA). All microarray specific reagents and device including hybridization oven and scanner were used from Agilent Technologies (Santa Clara, Calif., USA). The Agilent Feature Extraction Software Version 10.7.3.1 (Agilent Technologies, Santa Clara, Calif., USA) was used for feature extraction (protocol GE2_107_Sep09). Subsequent data analysis, including LOWESS normalization and statistical analysis were performed by use of the microarray and gene expression (MAGE)-compliant system EMMA 2 (Dondrup et al. 2009). A p-value of 0.05 was used as a cut-off for significance. The M-value cut-offs for a false discovery rate of 0.01 were determined as 1.1 and -1.1 according to previous “yellow experiments” performed by Wolf et al. (2017a).
Analysis of the Functional Relevance of Cgt
Distribution of Single-Domain CBM-20 Proteins in the Eubacterial World
The inventors have analyzed the distribution of CBM-20 single-domain proteins in the prokaryotic world by BlastP analysis.
In brief, the distribution of singular CBM-20-domain proteins was analyzed by BlastP analyses using the NCBI non-redundant protein database (Altschul et al. 2005; Altschul et al. 1990). As CBM-20 domains occur in a variety of different proteins and enzymes, data filtering had to be performed: Of the initial 3,316 BlastP hits, all of eukaryotic origin and all enzymes with function-specific annotation or sizes above 350 amino acids were excluded. The domain structures of the remaining 80 BlastP hits were analyzed (Marchler-Bauer et al. 2017; Marchler-Bauer and Bryant 2004; Marchler-Bauer et al. 2015; Marchler-Bauer et al. 2010). Most of these, 53 proteins in total, contain two CBM-20 domains traversed by a higher domain described as glyco-hydro-77-superfamiliy 4-alpha-glucanotransferase. Ten contain different additional domains: Five of them alpha-amylase inhibitor domains, two CBM-25, respectively, CBM-26 binding domains at the N-terminus, two N-terminal domains of IPT-superfamily with probable regulatory function and one a DUF1393-domain, which was described to occur in several alpha-amylases (information taken from the NCBI database). These candidates were also excluded. Only 18 candidates (including Cgt from Actinoplanes sp. SE50/110) displayed a singular CBM-20 domain. A protein tree was created by Blast tree view 1.17.5 of the NCBI database (NCBI database) on basis of a multiple sequence alignment performed by BlastP (Altschul et al. 1990; Altschul et al. 2005).
Interestingly, singular CBM-20 domain-proteins were found in only 17 other species (
Strains carrying singular CBM-20 proteins without direct connection to the habitats soil or environment occur only occasionally, like in singular isolates of the human pathogens Chlamydia trachomatis (Thomson et al. 2008) and Mycobacterium abscessus (Ryan and Byrd 2018; Moore and Frerichs 1953).
Confirmation of the Starch Binding Function by an In Vitro Assay
CBM-20 domains are described to have a starch binding function, which the inventors wanted to test by an in vitro assay. As the small carbohydrate binding protein Cgt is highly expressed and enriched in the extracellular space due to an N-terminal signal peptide (Wendler et al. 2015a), the protein could be directly concentrated from the supernatant by filtration. A starch binding assay was performed with starch from potato in different concentrations. Both—the starch fraction as well as the supernatant—were analyzed by SDS-PAGE. In all starch fractions (ranging from 1 to 10% (w/v) of starch), a protein band at about 15 kDA was detected, which was clearly identified as Cgt by MALDI-TOF-MS. In contrast, the supernatant fractions were almost completely depleted by Cgt. Residual Cgt in the supernatant was found, indicating, that the added starch was completely saturated by Cgt. In the negative control without starch, most of Cgt remains in the supernatant fraction. Beside Cgt, another small extracellular protein of unknown function, ACSP50_6253, was identified by the starch binding assay (data not shown).
Analysis of Cgt Expression During Growth on Different Carbon Sources
The gene cgt has been reported of being differentially expressed in the presence of different carbon sources, as determined by transcriptome and proteome analysis on glucose and maltose (Schwientek et al. 2013; Wendler et al. 2015a; Ortseifen 2016). The inventors have tested the effects of several carbon sources on the expression of cgt gene by measuring the transcript amounts by reverse transcription quantitative PCR (RT-qPCR). For this purpose, the wild type strain of Actinoplanes sp. SE50/110 was grown on minimal medium supplemented with maltose, glucose, starch, galactose, cellobiose, lactose and C-Pur (Cerestar 01908) (
For most tested carbon sources, the transcription of the cgt gene was similar or just slightly and insignificantly reduced compared to a maltose grown culture (
Analysis of Gene Deletion Mutant ΔCgt
ΔCgt on Different Carbon Sources or Under Carbon-Limited Conditions
The differential transcription profile of cgt in dependence of the carbon source indicated a function within sugar metabolism, like it has been presumed before (Ortseifen 2016). Ortseifen (2016) (Ortseifen 2016) suggested Cgt of being responsible for the retention of carbon as energy source in the context of the carbophore model. Growth of the wild type and the CRISPR/Cas9 deletion mutant Δcgt was tested on different carbon sources in liquid culture.
Before, a pre-screening experiment was performed in the OmniLog Phenotypic Microarray System (Biolog Inc., Hayward, United States of America), which allows fast phenotypic screening by measurement of cellular respiration activity on a total of 190 different carbon sources in multi-well plates. Of these, Actinoplanes displayed respiration on 103 carbon sources.
Except for arabinose and lactose, no differential respiration profile was observed for Δcgt on the remaining 101 carbon sources. In order to validate these results on the level of growth, the carbon sources arabinose and lactose were furthermore tested in a shake flask cultivation. Also, the standard laboratory sugars maltose and glucose, and the complex carbon source starch as well as the disaccharide cellobiose were tested, to imitate natural carbon sources of the habitat soil. No restraint on growth was observed for Δcgt (
Furthermore, growth under carbon-limited conditions (here: 1 g·L−1, 2 g·L−1, 3 g·L−1, 4 g·L−1 and 5 g·L−1 starch) was tested in the RoboLector®-system of m2p-labs. No growth disadvantages for the Δcgt mutant were observed in case of carbon source constraints compared to the wild type (
Δcgt has No Impact on Osmolarity- or pH-Tolerance Cgt multimers have been proposed to form surface layers through multimerization (Wendler et al. 2015a). This might suggest a potential role in protection against environmental changes, like drought, pH and osmolarity.
A pH screening was performed on solid media as well as in liquid culture in the RoboLector®-system. For screening on solid media, SFM-agar plates of pH ranging from pH 4 to 11 (in steps of 1) were prepared and droplets of a dilution series of spores of the wild type and the deletion mutant Δcgt were applied. Both mutant and wild type were able to grow from pH 5 to 11. No differences in growth or spore formation on agar-plates were observed.
Since an effect of drought tolerance is difficult to assess, the inventors analyzed the colony and spore formation on the surface of the bacterial lawn and found no differences between the wild type and Δcgt.
For pH screening in liquid culture, maltose minimal medium of pH ranging from 4 to 7 was prepared. Higher pH values could not be tested in liquid culture, as medium components tend to precipitate. Both strains grew from pH 4.5 to 7 (
For osmolarity screening, maltose minimal medium was prepared with different concentrations of maltose ranging from 3.6 to 108.1 g·L−1 maltose monohydrate and osmolarity ranging from 323.5 to 681.0 mOsmol·kg−1 (Table E11). No significant growth differences were observed between the wild type and the deletion mutant Δcgt (
Also, inositol was tested as osmolyte, since it is not consumed by Actinoplanes. Here, osmolarity ranged from 388.5 to 695.0 mOsmol·kg−1, but no growth differences were observed (
Lower osmolarities between 159-190 mOsmol·kg−1 were tested by use of the complex medium NBS (
Δcgt Displays Improved Acarbose Formation on Maltose Minimal Medium
Although no distinct growth phenotype could be observed under the tested conditions, lack of the highly expressed Cgt protein seems to save metabolic resources of the cell, such as ATP and amino acids. These might be used for cellular growth or other anabolic processes. In the experiments, Δcgt has not displayed significant growth advantages. However, remarkably higher final acarbose concentrations were detected for the deletion mutant Δcgt compared to the wild type (Table E10). For the cultivation in complex medium, this was most striking during the growth phase (
The improved acarbose-producing phenotype was validated by three independent shake flask cultivations in maltose minimal medium (
Findings that the deletion of the highly expressed gene cgt has no negative impact on growth or viability of the organism under various conditions, but yields into an enhanced acarbose producing phenotype, was surprising. Due to this and to rule out a direct impact on the regulation of acarbose biosynthesis (acb) genes, RT-qPCR of representative acb genes were performed. For this, wild type and Δcgt were grown on maltose-minimal medium and RNA was isolated from samples of the early growth phase. The relative transcript amount of the acarbose biosynthesis cluster genes acbZ, acbW, acbV, acbA, acbB, acbD and acbE were calculated for Δcgt in comparison to the wild type (
The connection of carbohydrate metabolism and acarbose biosynthesis is of high interest. Recent research has pointed out the importance of carbon utilization in the context of the biosynthesis of acarbose and further acarviosyl metabolites in the wild type (Wendler et al. 2014).
In this context, the starch binding protein Cgt is striking. It is one of the strongest expressed genes in Actinoplanes sp. SE50/110 (Schwientek et al. 2013) making up for about 8% of the whole secreted proteome (unpublished data of the inventors). Its gene product is exported into the extracellular space (Wendler et al. 2013). Excess production and export means high costs for the cell: Only for the translational process, 4 ATP are required per peptide bond (Campbell and Reece 2011; Purves 2006), i.e. not including additional costs for RNA synthesis, amino acid production, protein folding and export. The inventors therefore concluded that Cgt has a significant role in Actinoplanes sp. SE50/110 physiology. Two different functions of Cgt are proposed and analyzed herein: A role within the sugar metabolism and a role as surface protein.
Due to the starch binding domain Ortseifen (2016) (Ortseifen 2016) suggested, that Cgt might be involved in binding and retention of energy sources in the context of the carbophore model (Wehmeier 2003). Evidence was also given here by RT-qPCR, which displayed differential expression of the gene cgt in glucose-, galactose- and lactose-grown cultures compared to cultures grown on maltose, higher maltodextrins and cellobiose. This is in accordance with differential proteome analyses on the carbon sources maltose and glucose (Wendler et al. 2015a; Wendler et al. 2015b). These results indicate a carbon-dependent expression of cgt. It would be exciting to elucidate the regulatory mechanism. However, it remains to be considered that over 900 genes are putatively involved in transcriptional regulation in Actinoplanes sp. SE50/110, of which 697 are annotated as transcriptional regulators according to the annotation of Wolf et al. (2017b) (GenBank: LT827010.1).
A sugar-dependent expression of cgt might indicate a function within the utilization of maltose, higher maltodextrins and—potentially—also cellobiose. However, our studies of the deletion mutant Δcgt have not unveiled phenotypical differences regarding the carbon utilization. This was tested for a total of 105 different carbon sources, of which 103 were analyzed in the OmniLog screening system and six in liquid culture.
As the function of Cgt might be negligible under excess of carbon source but indispensable when growing under conditions with limited carbon source, the inventors have tested growth of the deletion mutant Δcgt and the wild type on minimal medium with low concentrations of starch.
Starch was chosen as carbon source, due to the starch binding activity of Cgt, which was confirmed in a starch binding assay here. Nevertheless, no growth phenotype of the mutant could be observed under limited carbon source conditions.
Another function within the sugar metabolism could consist in binding of insoluble crystalline substrates, which might lead to structural changes, that increases substrate accessibility and enhances the activity of other hydrolyzing enzymes like amylases. Such mechanisms have already been described in the soil bacteria Serratia marcescens for chitinolysis (Vaaje-Kolstad et al. 2005) and Thermobifida fusca for cellulysis (Moser et al. 2008). In the genome of Actinoplanes sp. SE50/110 several genes are encoded with putative α-glyosidic function, of which three, the α-amylases/pullulanases AcbE, AcbZ and PulA, were shown to accumulate in the extracellular space (Wendler et al. 2015a). Additionally, another small extracellular protein of unknown function and starch binding capability (ACSP50_6253) was identified in a starch binding assay. By heterologous expression of extracellular amylases and enzyme assays in presence and absence of both—Cgt and ACSP50_6253—, a supporting function during starch degradation might be detected in future experiments.
Apart from the sugar metabolism, also a function as surface layer protein is conceivable, which is supported by the fact, that Cgt forms multimers (Ortseifen 2016; Wendler et al. 2013). Wendler et al. (2015) (Wendler et al. 2015a) identified two transmembrane domains in the Cgt protein, of which one is involved in translocation by the Sec pathway as part of the leader peptide and the second is assumed to be required for multimerization. Although Cgt is not likely to be physically anchored in the membrane (Wendler et al. 2015a), Cgt proteins may remain as multimers in the mesh of the mycelium, due to the reduced fluid flow. In this context, the starch binding domain might serve also as anchor.
In the role as putative surface protein, the inventors initially assumed a protective function in the context of pH and osmolyte stress or drought. However, the screening experiments showed that the deletion of cgt gene did not lead to significant growth inhibition at different pH in liquid culture.
From the screening experiments on solid media, there was no indication, that Cgt might have a protective function in case of pH or drought.
Hints for a putative function in the context of osmoregulation were given by reverse transcription quantitative PCR of the wild type, grown on different amounts of maltose. Here, the inventors observed a 2.9-fold reduced transcription of the gene cgt, when growing on 44.4 g·L−1 maltose compared to a 72 g·L−1, which might be an effect of osmolarity. The inventors analyzed growth of the deletion mutant Δcgt in several screening experiments in liquid culture with media ranging from 159 to 681 mOsmol·kg−1. Under all tested conditions, no differences in growth and viability were observed for the deletion mutant Δcgt compared to the wild type.
As surprisingly no apparent physiological impact was observed by the deletion of cgt gene neither in utilization of different carbon sources in excess nor in limitation, neither under different pH nor osmolyte conditions, it might be possible that the function of Cgt only becomes apparent in its natural environment and in possible competition with other soil organisms. Interestingly, the inventors found similar independent singular CBM-20 domain proteins in 17 other prokaryotic species, most of which belong to the order Actinomycetales. Although rare, this at least displays a certain distribution and shows, that Cgt is not a strain-specific protein. Most of the species harboring single domain CBM-20 proteins were associated with soil habitats. Together with the fact, that cgt is highly expressed in Actinoplanes sp. SE50/110, this supports the hypothesis that proteins like Cgt fulfill a crucial function in bacteria living within this habitat. A function of Cgt could be tested in future by co-cultivations in direct contact with other microbial competitors.
While it was surprising, that Cgt turned out to be dispensable under the tested laboratory conditions, the inventors observed a positive phenotype regarding the acarbose production. An increase of the acarbose yield between 8.3 and 16.6% was achieved by deletion of cgt. Although the final product yields differ slightly between batch cultivation, the Δcgt mutant always performed significantly better. This was shown over a time period of several month (data not shown) in three independent shake flask and several micro-scale cultivations performed in maltose minimal medium. Thus, the improved production was robust over long time periods and in different cultivation settings.
We assume that this is due to metabolic burden by expression of cgt gene in the wild type, which brings relief of energy and of free resources in Δcgt. These resources are probably redirected to the acarbose biosynthesis, which is a growth-associated product. A direct regulatory effect by deletion of cgt on the expression of the acb genes was not observed.
Analysis of the Functional Relevance of Carotenoid Formation
Light-Dependent Carotenoid-Formation and Oxidative Stress Reduce Acarbose Production in Actinoplanes sp. SE50/110
Actinoplanes are known to produce a variety of soluble pigments including yellow, orange and pink pigments of the class carotenoids (Parenti and Coronelli 1979). The pigment of Actinoplanes sp. SE50/110 is orange. Its formation is intensified when cultivated exposed to light. Since the pigment was found likewise in the supernatant, it seems to be soluble in watery solutions. After cell extraction and separation by thin layer chromatography, spectral analysis display absorption maxima at 450, 475 und 505-510, which was confirmed by an absorbance scan performed during HPLC-separation. Consistent with these findings in silico reconstruction shows, that Actinoplanes sp. SE50/110 has the full genetic equipment to produce a C40-carotenoid with similarity to sioxanthin from Salinospora tropica CNB-440 (Richter et al. 2015; Wolf et al. 2017b) (
indicates data missing or illegible when filed
The genes of the C40-carotenoid biosynthesis are organized in three gene cluster: terpene cluster 1, 2a and 2b (cf.
In contrast to S. tropica, homologues of crtY and crtU, encoding a cyclase and a desaturase, could not be identified in Actinoplanes sp. SE50/110 (Wolf et al. 2017b). Instead, two cyclases of the CarR-domain superfamily were found in this work. They are localized in the terpene cluster 2b (
Comparative genome analysis by the software platform EDGAR 2.0 (Blom et al. 2016), display similar terpene cluster arrangements in related species of the genus Actinoplanes, whereas a different organization was found in Streptomyces (data not shown). By this, the gene arrangements found in SE50/110 and CNB-440 (Richter et al. 2015; Wolf et al. 2017b) seem to be characteristic for the family Micromonosporaceae.
Besides, genes for the synthesis of the building blocks IPP and DMAPP via the MEP/DOXP-pathway (Table E12), a gene coding for a camphene-like monoterpene synthase (terpene cluster 3, Table E12) as well as a carotenoid cleavage dioxygenase (ACSP50_5522, Table E12) were found in the genome of SE50/110. The latter two might be involved in the formation of odorous substances (Yamada et al. 2015). The inventors observed, that strong pigmentation was associated with production losses. This was confirmed by comparing growth and acarbose yields of cultures exposed to and covered from light (
Deletion of merR in SE50/110 Induces Carotenoid Formation without Exposure to Light
Since natural or bulb light was able to induce carotenoid formation (
The MerR-family mainly consists of activators, which are able to respond to environmental stimuli, like oxidative stress, heavy metals or antibiotics (Brown et al. 2003). Indeed, several members of the MerR-family have been described as both light-dependent activators or repressors of the carotenoid biosynthesis in non-photosynthetic bacteria, f. e. LitR in the related actinomycete S. coelicolor (Takano et al. 2005; Takano et al. 2006), in the Gram-negative Thermus thermophiles HB27 (Takano et al. 2011) and in the Gram-positive Bacillus megaterium QM B1551 (Takano et al. 2015). Here, cobalamin (vitamin B12) acts as cofactor, which mediates light sensitivity, since it is able to absorb ultraviolet and blue light: By either binding covalently to the regulator or falling off after light excitation, it is able to modulate the conformation and activity of the regulator (van der Horst et al. 2007). The mechanisms of regulation and the binding sites are quite different: Whereas in T. thermophiles and B. megaterium the promoter regions of litR/crtB (Takano et al. 2011) or litR and crtl (Takano et al. 2015) are repressed in the dark and relieved after illumination, LitR in S. coelicolor seems to be an essential light-induced transcriptional activator of the adjacent localized litS, which encodes an ECF sigma factor and directs the transcription of the carotenoid biosynthesis genes (Takano et al. 2005). A gene encoding an ECF sigma factor does not occur within the gene cluster of SE50/110. In the Gram-negative bacterium Myxococcus xanthus a B12-dependent MerR regulator is part of a complex regulatory cascade including eight further regulatory genes (Fontes et al. 2003; Galbis-Martinez et al. 2012). Indeed, no homologues of the regulatory network from M. xanthus were identified in the genome of SE50/110 by BLASTP-analysis (data not shown).
The MerR-family regulator ACSP50_0145 of Actinoplanes sp. SE50/110 contains an N-terminal HTH-motif and a C-terminal B12-binding domain (according to BLASTP-analysis and CDD-search (Marchler-Bauer et al. 2015; Marchler-Bauer et al. 2010; Altschul et al. 2005)). The position of the HTH-domain accounts for a transcriptional repressor (Pérez-Rueda and Collado-Vides 2000).
By CRISPR/Cas9 deletion of the corresponding gene in SE50/110, the carotenoid formation was strongly induced without exposure to light (
Indeed, it has to be noted, that the repressor/operator system is leaky, since the typical orange color is also produced in the wild type without exposure to light. According to this, the transcription of the genes crtEBI and idi (ACSP50_0146-0149) was only doubled in AmerR compared to the wild type under dark conditions (
However, in the context of this work, the question was examined, whether pigment formation in AmerR influences the formation of the fine-chemical acarbose. Again, higher carotenoid formation was associated with lower acarbose formation (
Under dark conditions, AmerR produces approximately 15% less acarbose than the wild type (0.70 g·L−1) (
Comparative transcriptome analyses of the wild type cultivated under dark and light conditions using the microarray technique, display a complex response on transcript level affecting various genes (cf.
In SE50/110, the tyrosinase MeIC (ACSP50_4950, previously: ACPL_5017), a photo-protector, which is involved in the formation of the brown pigment eumelanin (Wolf et al. 2016), and genes of the riboflavin biosynthesis (ACSP50_6437-40) are stronger transcribed when exposed to light (
According to this, also several flavin-dependent oxygenases are stronger transcribed, when exposed to light. One of them is annotated as taurine dioxygenase, which substrate is a degradation product of cysteine. Sulfur-containing amino acids like cysteine belong to the group of low molecular weight thiols (LMW thiols), that are able to catch ROS and function as redox buffers (Gout 2019). Corresponding to this, further genes probably involved in cysteine and methionine metabolism and transport are stronger transcribed in cells exposed to light.
Remarkably, several transcriptional regulator genes and a gene encoding the sigma factor SigE (ACSP50_0558) are stronger transcribed, too (
Interestingly, genes of the carotenoid biosynthesis and of the regulator MerR are not significantly stronger transcribed in the wild type exposed to light compared to the wild type hidden from light. This is noteworthy, since a clear effect of light on carotenoid formation can be observed in the wild type. Since the carotenoid synthesis takes place both in the dark and in the light and the enhancement of relative transcript amounts is quite moderate in the regulator mutant (see above), the effects on transcript level might be inconspicuous. It is assumed, that further regulation of carotenoid synthesis on protein level or metabolome level might exists, f. e. by degradation of carotenoids or terpenoid-precursors by the carotenoid cleavage dioxygenase (ACSP50_5522). However, according to the results obtained from the microarray of the wild type, the crt gene expression does not seem to be a primary target of the global oxidative stress response, similar to findings from Rhodococcus sphaeroides (reviewed in Ziegelhoffer and Donohue (2009)).
Taken all together, illumination triggers oxidative stress response and seems to have an important impact on the distribution of metabolic resources towards growth, carotenoid and acarbose formation. The regulation of carotenoid biosynthesis seems to be decoupled from the global response to oxidative stress, which needs further investigation. With view to direct the metabolic fluxes towards the production of acarbose, it is desirable to gain a better understanding of these processes in future. The sigma factor SigE might be responsible for the oxidative stress response, since it is higher transcribed when exposed to light.
Apart from light stress, this work demonstrates, that a large portion of production losses can be directly assigned to the carotenoid formation. Carotenoids of non-photosynthetic bacteria are assumed to have a function as photo-protectors (Lee and Schmidt-Dannert 2002), since they have shown to protect from photodynamic killing (Mathews and Sistrom 1959). As the influence of light can be excluded by simple structural measures, carotenoid formation is assumed to be dispensable under laboratory conditions. In order to improve acarbose production, switching off the concurring carotenoid biosynthesis pathway, f. e. by deletion of the central gene crtl, can be used for strain development. Since carotenoids influence the fluidity of membranes (Gruszecki and Strzalka 2005), lack of the C40-carotenoid can also affect the surface and mycelial structure of Actinoplanes sp. SE50/110. With regard to production, a break-up of mycelial lumps is advantageous to increase the mycelial surface and the number of biochemically available cells.
Overexpression of acbB and gtaB
Expression vector pSETT4 was tested for the genes acbB and gtaB. Both genes, acbB and gtaB, are probably involved in the amino sugar synthesis, a feeding branch of acarbose biosynthesis: AcbB catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose and GtaB is assumed to be involved in the supply of the precursor glucose-1P. Interestingly, both proteins display increased protein amounts in the cytosol of acarbose producer.
pSETT4gap and pSETT4tip Vectors for Overexpression of Single Genes
A novel cloning system was implemented, that allows easy cloning and overexpression of singular genes in Actinoplanes strains such as Actinoplanes sp. SE50/110. For this, the strong promoter of the gene gapDH from Eggerthella lenta was cloned in front of a lacZ-cassette in a pSET152-backbone. The gene lacZ is transcribed under control of the lac-promoter and flanked by the recognition side of the restriction enzyme BsaI, which enables exchange of lacZ by the gene of interest by Gibson Assembly (Gibson et al. 2009), restriction/ligation cloning or Golden Gate cloning (Engler et al. 2008). As strong expression requires strong termination, T4-terminators were introduced before and after the cloning side of the novel expression system. T4-terminators have already been successfully used in the pGUS-cloning system developed by Myronovskyi et al. (2011). Whole track RNAseq analysis of a pGUS-integration mutant performed herein showed, that the T4-terminators block transcription efficiently and prevent read-through from the integrase gene into the gene of interest. Like shown by a pre-experiment, T4-terminators do not have any side effects on the transcription of acb genes, when introduced into Actinoplanes sp. SE50/110 via pSET152-integration.
Besides, by sequencing of an enriched primary transcript library derived from the promoter-screening experiment, two putative promoters were identified behind the gene of interest in antisense orientation (
To allow exchange of the promoter sequence, NdeI and KpnI restriction sites were introduced. In this work, the strong gapDH-promoter was exchanged by the medium-strong tipA-promoter from S. lividans. By this, it was shown, that the system can be easily modified, f. e. to adjust it for other species of the order Actinomycetales. The vectors (named pSETT4gap and pSETT4tip) were tested for strong and medium strong overexpression of the genes acbB and gtaB.
Medium Overexpression of acbB Leads to Improved Acarbose Formation
The dTDP-D-glucose-4,6-dehydratase AcbB seems to be involved in the generation of an activated amino sugar from D-glucose-1P—a feeding pathway of the acarbose biosynthesis (
The mutant with acbB transcribed under control of the heterologous tipA-promoter displayed enhanced acarbose production compared to the control strains: The yield coefficient was increased to 48.6 and 51.9% compared to the empty vector control in two independent cultivations (
In pSETT4tip::acbB, the normalized peak areas of phosphorylated glucose/galactose and UDP-glucose were similar or even slightly increased compared to the empty vector control (
Therefore, the supply of activated glucose moieties seems to be guaranteed. In this mutant, increased amounts of the mass m/z=545 [M-H+] were found (
At beginning growth phase enhanced expression of acbB was observed in the expected range: Strongest overexpression was achieved by use of the gapDH-promoter (log2(fold-change)=6.54) followed by use of the tipA-promoter (log2(fold-change)=4.06) (
This was tested in both the pSET152- and pSETT4-vector background (
Only exception is a slightly higher transcription abundance of acbA in pSETT4tip::acbB (log2(fold-change)=1.87).
Remarkably, the transcription profile in the linear growth phase differs from the early growth phase: Here, only a doubling of transcript amounts was reached by use of the gapDH-promoter (log2(fold-change)=2.05), whereas by use of the tipA-promoter the overexpression of acbB was maintained, but to a lesser extent (log2(fold-change)=3.33) (
In overexpression mutants including heterologous promoters the relative transcription of acbB decreases from 4.06- to 3.33-fold (log2(fold-change)) between the two sampling times in pSETT4tip::acbB and from 6.54- to 2.05-fold in in pSETT4gap::acbB. It is assumed, that whereas the transcription of the chromosomal acbB-copy is down-regulated in these mutants, the transcription of the vector copy is maintained by the heterologous promoters. The differences in acbB-transcription at different sampling times furthermore suggest, that the down-regulation of acb gene transcription occurs stronger respectively earlier in pSETT4gap::acbB compared to pSETT4tip::acbB. Overexpression of acbB (pSETT4gap::acbB and pSETT4tip::acbB) seems to decelerate during linear growth phase.
In summary, in particular medium overexpression of acbB by usage of the tipA-promoter seems to be beneficial for acarbose production, whereas strong overexpression by use of the gapDH-promoter seems to have only a smaller effect on acarbose formation. Further improvement in acarbose formation may be achieved by varying of the expression level of acbB, e.g. by using alternative promoters from the promoter screening or by introducing multiple gene copies.
In summary, this work demonstrates, that medium overexpression of AcbB increases the acarbose yields, possibly due to improved amino sugar supply. By medium overexpression of acbB (e.g. by use of the tipA-promoter), a positive effect on acarbose production was observed yielding into round about 50% more acarbose in two independent cultivations. Therefore, the improvement of the acarbose biosynthesis by overexpression of singular acb genes was achieved.
Medium Overexpression of gtaB Leads to Improved Acarbose Formation
GtaB is supposed to catalyze the conversion of UDP-glucose and glucose-1P into each other. It was surprisingly found that overexpression of GtaB triggers acarbose formation. Without being bound by theory this may occur by improved deployment of the precursor glucose-1P. As shown by a shake flask cultivation in maltose minimal medium (
Interestingly, the acarbose formation is particularly increased in the late linear to stationary growth phase. In the overexpression mutant, the relative transcript amount of the gene gtaB is 2.64-fold increased (log2(fold-change)) (
Since the metabolism of activated sugars is connected or redirected to other metabolic pathways, they are not supposed to accumulate. But—like shown in previous experiments—the supply can be seriously disturbed. Analysis of the intracellular metabolome displays similar amounts of phosphorylated hexoses and/or UDP-glucose (
Interestingly, a significant decreased amount of the mass m/z=545 [M-H+] was found in pSETT4tip::gtaB (approx. decrease of 48%), which might correspond to dTDP-4-keto-6-deoxy-D-glucose, the proposed product of AcbB. This may indicate, that the flow through the synthesis strand is more balanced, since the accumulation of this metabolite is reduced in comparison to the empty vector control and AcbB-overexpression mutants (
Transfer of this construct to producer strains of Actinoplanes can result in an increase of the beneficial effect, as here the demand for the precursor is higher compared to the wild type. Since strong overexpression of AcbB leads to an imbalance in glucose-phosphate-metabolism combined overexpression of acbB and gtaB would plausibly further improve acarbose production beyond the observed effect for the single overexpressions.
Krause, Jens P., Tino Polen, Jung-Won Youn, Denise Emer, Bernhard J. Eikmanns, and Volker F. Wendisch. “Regulation of the malic enzyme gene malE by the transcriptional regulator MalR in Corynebacterium glutamicum.” Journal of biotechnology, 2012: 204-215.
Woo, Han Min, Stephan Noack, Gerd M. Seibold, Sabine Willbold, Bernhard J. Eikmanns, and Michael Bott. “Link between phosphate starvation and glycogen metabolism in Corynebacterium glutamicum, revealed by metabolomics.” Applied and environmental microbiology, 2010: 6910-6919.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19203693.7 | Oct 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/077688 | 10/2/2020 | WO |
