Improved Method for Forming Acarbose

Abstract

The present invention relates to actinomycete strains for improved acarbose formation. Actinomycete strains engineered to overexpress dTDP-D-glucose-4,6-dehydratase (AcbB) and / or uridyltransferase (GtaB) are provided. Also provided are actinomycete strains engineered to have reduced or eliminated expression of small carbohydrate-binding proteins (Cgt) and / or reduced or eliminated expression of genes essential for carotenoid synthesis. Tools, methods, and means for generating these strains are also provided.

Description

Technical Field

[0001] Field of Disclosure The present invention relates to Actinomycetales strains for the improved formation of acarbose. Provided are Actinomyces strains engineered to overexpress dTDP-D-glucose-4,6-dehydratase (AcbB) and / or uridyltransferase (GtaB). Also provided are Actinomyces strains engineered to have a decrease or absence in the expression of low molecular weight carbohydrate-binding protein (Cgt) and / or a decrease or absence in the expression of genes essential for carotenoid synthesis. Also provided are tools, methods, and means for generating these strains.

Background Art

[0002] Background Acarbose The therapeutic agent acarviosyl-maltose (acarbose) has been used since 1990 in the medical treatment of diabetes (Wehmeier and Piepersberg 2004; Wehmeier 2004). It is intended to assist the patient's strict diet plan and prevent sugar peaks during high carbohydrate intake. After oral administration, acarbose inhibits intestinal α-glucosidase and delays the release of monosaccharides from starch- and sucrose-containing diets. Thereby, acarbose helps control the rate of absorption of monosaccharides into the bloodstream and results in a decrease in postprandial blood glucose and serum glucose levels, which are assumed to be extremely important in relation to cardiovascular disease mortality.

[0003] Industrial Applicability Acarbose is known as Glucobay (Bayer AG) in Europe and China, Precose (Bayer Pharmaceuticals) in North America, and Prandase (Bayer AG) in Canada, and is commercially available. Since it is an important and highly demanded drug for the treatment of diabetes, it is necessary to provide high-yield and high-quality acarbose. As the incidence of type II diabetes is continuously increasing worldwide, the optimization of product yield and quality is a current concern.

[0004] Acarbose-producing strain Acarbose is naturally produced by different Actinomycetales, such as 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), the latter being the wild type of industrial production strains (Ortseifen 2016; Mahmud et al., 1999). The genus Actinoplanes was first introduced by Couch (1950) as a member of the family Micromonosporaceae, order Actinomycetales, phylum Actinobacteria. Actinoplanes sp SE50 / 110 (ATCC 31044, CBS 674.73) is a slow-growing natural derivative of SE50 (ATCC 31042, CBS 961.70) (Frommer et al., 1973). SE50 was isolated in 1970 during a screening program by Bayer AG from soil samples near a coffee plantation in Kenya (Frommer et al., 1972). SE50 / 110 produces approximately 1 g·L when maltose is provided in the medium -1producing acarbose (Wendler et al., 2014). Further producer strains are engineered as described, for example, in (EP2601209B1) and (CN103298828B).

[0005] For Actinoplanes sp. SE50 / 110, it has been shown that the biosynthesis of acarbiosyl-sugars depends on the supply of a carbon source in the culture medium (Wendler et al., 2014). When growing on glucose, acarbiosyl-glucose was formed as the main compound, but when growing on maltose (Wendler et al., 2014), mainly acarbiosyl-maltose was formed, and when growing on maltotriose (Ortseifen 2016), acarbiosyl-maltotriose was formed.

[0006] For medical and industrial relevance as the wild type of industrial acarbose producer strains, Actinoplanes sp. SE50 / 110 has been extensively studied in recent 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) have been comprehensively analyzed. Thereby, a precise genomic sequence was obtained and the annotation was improved in 2017 (GenBank: LT827010.1) (Wolf et al., 2017b). Also, an intergeneric conjugation system (Gren et al., 2016) has been established, similar to advanced genome editing tools using CRISPR / Cas9 (Wolf et al., 2016), enabling targeted genetic engineering. Nevertheless, for Actinoplanes sp. SE50 / 110, a reliable expression system enabling moderate to strong gene expression is lacking. Since there was no system suitable for moderate overexpression of specific genes before, different strategies were tested and evaluated according to the present invention, and a new expression system called pSETT4 was developed.

[0007] Acarbose biosynthesis The acarbose biosynthetic pathway is based on single-functional enzymes that catalyze a single step (Figure 1) (Wehmeier and Piepersberg 2009). According to the model of Zhang et al. (2002), biosynthesis proceeds via the intermediate valienone-7P. In an improvement by Wehmeier (2003), the stepwise reduction and dehydration changed, and valiolol-7P was obtained as an intermediate. Since the order of the steps is unknown, they are shown in parentheses. The first step of acarbose biosynthesis, which is a cyclic reaction by AcbC to form 2-epi-5-epi-valiolone from sedo-heptulose-7P, is missing in this figure. Although the focus of research for the past few decades, the acarbose biosynthetic pathway has not yet been fully elucidated. Only the first three steps of biosynthesis have been experimentally confirmed. AcbC (ACSP50_3607), the first enzyme in acarbose biosynthesis, catalyzes a cyclic reaction to produce 2-epi-5-epi-valiolone from sedoheptulose 7P7P (Stratmann et al., 1999). Phosphorylation to 2-epi-5-epi-valiolone-7P is catalyzed by the kinase AcbM (ACSP50_3603) in the presence of ATP (Zhang et al., 2002), and epimerization to 5-epi-valiolone-7P is catalyzed by the cofactor-independent epimerase AcbO (ACSP50_3606) (Zhang et al., 2002; Zhang et al., 2003).

[0008] Remaining steps of the model based on protein homology and function prediction (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 cyclitol dehydrogenase / oxidoreductase AcbN (ACSP50_3605) are suggested to catalyze the reduction of 1-epi-valienol-7P and 5,6 dehydration. Phosphorylation to 1,7-diphospho-1-epi-valienol is thought to be catalyzed by 1-epi-valienol-7-phosphate-1-kinase AcbU (ACSP50_3595) and / or hydrolase AcbJ (ACSP50_3600). Nucleotidylation to NDP-1-epi-valienol-7P is probably catalyzed by GlgC-related NDP-polyol synthase AcbR (ACSP50_3597) (1-epi-valienol-1,7-bisphosphate-1-adenylyltransferase), and the transfer of the activated intermediate to the activated amino sugar is mediated by glycosyltransferase AcbI (ACSP50_3599) and / or AcbS (ACSP50_3596) to produce akalivosin-7P.

[0009] Activated amino sugars are thought to be synthesized in three steps from D-glucose-1-phosphate (Wehmeier and Piepersberg 2004; Wehmeier and Piepersberg 2009; Zhang et al., 2002), which are: (i) nucleotide addition of dTDP-D-glucose by dTDP-glucose-synthase AcbA (ACSP50_3609), (ii) dehydration to dTDP-4-keto-6-deoxy-D-glucose by dTDP-D-glucose-4,6-dehydratase AcbB (ACSP50_3608), and (iii) amination to dTDP-4-amino-4,6-dideoxy-D-glucose by GabT-like aminotransferase AcbV (ACSP50_3594) (Diaz-Guardamino Uribe 2000; Zhang et al., 2019).

[0010] Glucose-1P is a branched metabolite that plays an important role in different pathways, such as glycogen metabolism, galactose metabolism, and glycolysis after conversion to glucose-6P (Frey 1996; Purves 2006). UDP-glucose-1P uridylyltransferase GtaB catalyzes the reaction that interconverts glucose-1P and UDP-glucose.

[0011] Finally, maltose is potentially transferred in a one-step reaction by AcbS (Hemker et al., 2001). However, AcbI or AcbJ have also been proposed to catalyze the transfer reaction (Wehmeier and Piepersberg 2004; Wendler et al., 2013). Another candidate for this reaction could be amylomaltase AcbQ (ACSP50_3601).

[0012] In Actinoplanes sp. SE50 / 110, the biosynthetic genes are organized in the acarbose biosynthetic 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 (Figure 2).

[0013] In addition to the biosynthetic genes (acbCMOLNUJRSIVBA) already mentioned, the cluster encodes functions in extracellular starch degradation (AcbEZ, ACSP50_3610 and ACSP50_3590), transglycosylation (AcbD, ACSP50_3611) and acarbose export (AcbWXY, ACSP50_3591-3). Furthermore, acarbose-7-kinase (AcbK, ACSP50_3602) and intracellular amylomaltase (AcbQ) are encoded, which have been assigned functions within the carbophore (Wendler et al., 2015b; Schwientek et al., 2012; Wehmeier and Piepersberg, 2009). The function of AcbP (ACSP50_3598), annotated as an NTP-pyrophosphohydrolase, is unknown.

[0014] Potentially metabolically relevant Actinoplanes proteins The specific CBM-20 domain protein Cgt is one of the most strongly expressed genes in the actinomycete species SE50 / 110 and the induced acarbose-producing strain (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 constitutes 8% of the total secretome of this organism (data not shown). Cgt contains 149 amino acids and a CBM-20 domain of fold family 1, functional group A, and is characterized by a β-sandwich structure (Schwientek et al., 2013; Guillen et al., 2010 [where the 'e' in 'Guillen' is correctly the letter 'e' with an acute accent]). Members of this family have been described to bind to starch (Guillen et al., 2010).

[0015] Methods for gene deletion in Actinoplanes The establishment of intergeneric conjugation systems (Gren et al., 2016) and CRISPR / Cas9 technology (Wolf et al., 2016) enables genome editing in the actinomycete species SE50 / 110.

[0016] Furthermore, according to the present invention, the inventors successfully established a novel deletion system by homologous recombination using an integrase-free vector backbone and CodA for counterselection as described by Zhao et al. (2017). This further expanded the genetic toolbox of the genus Actinoplanes species SE50 / 110. As proof of principle, the novel deletion system was successfully tested for the deletion of the example gene cgt. Homologous recombination (HR) is a common process in actinomycetes and can be technically used to create deletion mutants by double crossover. Temperature-sensitive replicons, similar to the pSG5 replicon, can support and enforce this process. (Du et al., 2015; Garg and Parry, 2010; Myronovskyy et al., 2009; Zhang and Parry, 2007). Additional methods, such as CRISPR-base editing systems for single nucleotide exchange, CRISPR-BEST by Tong et al., 2019, CRISPRi / dCas9 by Qi et al., 2013, RNA interference, etc. exist in the art.

[0017] Methods for gene overexpression in Actinoplanes Actinoplanes species SE50 / 110 has been extensively studied over the past few decades. Designing an appropriate expression system is difficult (see Schaffert et al. (2019)). The entire content of the publications, particularly the description of expression systems and promoters for genetic manipulation of Actinoplanes, are hereby incorporated by reference in their entirety.

[0018] Previous studies have shown successful gene expression by using pKC1139 in A. teichomyceticus (Horbal et al., 2012). However, the replicative pSG5-based vector pKC1139 (constructed by Bierman et al. (1992)) has been found to be unsuitable for the expression of homologous genes in Actinoplanes sp. SE50 / 110 because unwanted vector integration by homologous recombination occurs (see Schaffert et al., (2019)). This is presumably due to the high metabolic cost of vector replication and is considered a preferred process. Without being bound by theory, the protein encoded by ACSP50_7170 in SE50 / 110 and predicted as recombinase A (recA) may catalyze the recombination process. Interestingly, no homolog of recA was found in the genome of A. teichomyceticus. The presence of recA in a. sp. SE50 / 110 and the deletion in A. teichomyceticus provide a definitive explanation for why HR-mediated vector integration has not been previously reported for A. teichomyceticus. Therefore, the deletion of the recombinase gene recA in Actinoplanes sp. SE50 / 110 may allow for the implementation of a pSG5-based replicative expression system.

[0019] Other replicative Streptomyces-E. coli shuttle plasmids, such as pKC1218 (which is based on the SCP2* replicon (Kieser et al., 2000)) and pSOK101 (which is based on the pIJ101 replicon (Zotchev et al., 2000)), did not give rise to conjugation-competent bodies with Actinoplanes sp. SE50 / 110 (Gren, 2017). These replicons are presumably unstable or inactive in SE50 / 110, which is consistent with findings from the related species A. teichomyceticus (Horbal et al., 2012).

[0020] By using an integrative vector system, genetic duplication can be achieved by integrating complete vectors with additional gene copies at different positions on the genome. This process is mediated by phage integrase. Phage integrase catalyzes the targeted unidirectional recombination of two attachment sites, attP located on the plasmid and attB located on the host chromosome (te Poele et al., 2008). After integration, the vector is flanked by the left (attL) and right (attR) attachment sites resulting from attP-attB recombination (te Poele et al., 2008).

[0021] Four different integrative vector systems have been described for Actinoplanes sp. SE50 / 110 (Gren et al., 2016): two are based on the integration mechanism of phage φC31 (pSET152 and pIJ6902). Vectors pRT801 / 2 and pSOK804 are based on the integration mechanisms of phage φBT1 and VWB phage. However, doubling of the relative transcription level by using natural promoters was not achieved (see Schaffert et al. (2019)).

[0022] Evaluation of homologous and heterologous promoters for integrative vectors A method for evaluating homologous and heterologous promoters with respect to their strength was provided by Schaffert et al. (2019), which is incorporated herein in its entirety. Briefly, 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 at the protein level, and 12 of these were analyzed at the transcriptional level (Table 1, Figure 3).

[0023]

Table 1

SUMMARY OF THE INVENTION

[0024] Strategy For the present invention, acarbiosyl-maltose metabolism was studied by gene deletion and overexpression, leading to a set of related tools and methods for engineering strains for improved production of acarbose. To improve acarbose synthesis, three different strategies were followed: (i) increasing the gene dosage of the acb gene to enhance the flux through acarbose biosynthesis, (ii) developing precursors for acarbose biosynthesis, and (iii) reducing the metabolic burden (Figure 4). The approach for each of these related strategies surprisingly resulted in improved acarbose formation: by overexpressing dTDP-D-glucose-4,6-dehydratase AcbB, the final acarbose concentration increased significantly by about 50%. Overexpression of uridyl transferase GtaB improved the acarbose yield by 8.5%, probably due to improved supply of the precursor glucose-1P. Functional deletion of the small carbohydrate-binding protein Cgt significantly enhanced acarbose formation by 8-16%, probably because the metabolic burden was thereby reduced. The enhancement was robust over a long period and in different culture environments. Furthermore, growth experiments of wild-type and regulator mutant ΔmerR exposed to and hidden from light were performed to clarify the negative effects of photoinduced stress and carotenoid formation on acarbose production. As a result, acarbose production can be further improved by reducing carotenoid formation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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[0026] Brief description of sequence IDs The sequence listing related to this application is submitted in electronic format and is hereby incorporated by reference in its entirety into this specification.

[0027] [Table 2] TIFF0007711052000005.tif254165TIFF0007711052000006.tif254166TIFF0007711052000007.tif254166TIFF0007711052000008.tif254166TIFF0007711052000009.tif254166TIFF0007711052000010.tif254165TIFF0007711052000011.tif254165TIFF0007711052000012.tif254165TIFF0007711052000013.tif254165TIFF0007711052000014.tif254165TIFF0007711052000015.tif254166TIFF0007711052000016.tif254165TIFF0007711052000017.tif251166TIFF0007711052000018.tif254165TIFF0007711052000019.tif246167TIFF0007711052000020.tif254165TIFF0007711052000021.tif254165TIFF0007711052000022.tif254165TIFF0007711052000023.tif254165TIFF0007711052000024.tif254164TIFF0007711052000025.tif254165TIFF0007711052000026.tif254165TIFF0007711052000027.tif254165TIFF0007711052000028.tif252167TIFF0007711052000029.tif254165TIFF0007711052000030.tif254165TIFF0007711052000031.tif254165TIFF0007711052000032.tif254165TIFF0007711052000033.tif254165TIFF0007711052000034.tif254165TIFF0007711052000035.tif254165TIFF0007711052000036.tif254165TIFF0007711052000037.tif254166TIFF0007711052000038.tif254166TIFF0007711052000039.tif254165TIFF0007711052000040.tif251166TIFF0007711052000041.tif254166TIFF0007711052000042.tif254166TIFF0007711052000043.tif254166TIFF0007711052000044.tif59167.

[0028] Detailed Description Definitions Unless otherwise defined, all scientific and technical terms used in the description, drawings, and claims have their ordinary meanings as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. If two or more documents incorporated by reference conflict with and / or contain conflicting disclosures, the document having the later effective date shall govern. Materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise specified, the following terms used in this specification, including the claims, have the definitions given below.

[0029] Terms such as "comprising", "including", "containing", "having", etc. shall be read expansively, or read with an open end and without limitation. Singular forms such as "a", "an", or "the" include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, the term "at least" preceding a series of elements shall be understood to refer to all elements of the series. The terms "at least one" and "at least one of" include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more elements.

[0030] It should be understood that using slight variations above and below the stated ranges can achieve substantially the same results as the values within the ranges. Also, unless otherwise specified, the disclosure of a range is intended to be a continuous range that includes every value between the minimum and maximum values.

[0031] Throughout this application, when a protein or amino acid sequence is provided, it is also understood by those skilled in the art that single or multiple amino acids can be replaced by amino acids with similar properties to achieve substantially the same effect, i.e., equivalent results. Those skilled in the art also know that a defined protein or amino acid sequence can be encoded by various nucleic acid sequences. For a given amino acid sequence defined herein, each of the countable nucleic acid sequences encoding the specific amino acid sequence is considered to be disclosed herein. When a nucleic acid sequence is provided throughout this application, it is further understood that silent mutations can be introduced.

[0032] O-{4,6-dideoxy-4[1S-(1,4,6 / 5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino-α-D-glucopyranosyl}-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose or "acarbose" is a cycloitol-containing aminoglycoside consisting of a pseudodisaccharide and an α-1,4-glycosidic bond maltose (Wehmeier and Piepersberg, 2009). The pseudodisaccharide called acarviose is constructed by an unsaturated C7-aminocyclitol also called valienol or valienamine, which is linked to C4 of 4,6-dideoxy-D-glucose by a nitrogen bond (see Figure 5) (Wehmeier and Piepersberg, 2009). This N-glycosidic bond cannot be hydrolyzed by exogenous α-1,4-glucosidase and results in an almost irreversible inhibitory effect (Wehmeier and Piepersberg, 2009; Brayer et al., 2000).

[0033] "Overexpression" of a gene product or protein as described herein refers to an increase in expression compared to wild type or a specific reference strain. Preferably, the reference strain or control is a strain that has not been engineered for specific overexpression of the respective gene or protein. For example, the control does not contain a vector comprising an expression cassette for the respective gene product or protein. For example, overexpression of a gene product can be an increase during the initial growth phase, during the linear growth phase, during the stationary phase, or during any other time. Preferably, overexpression is an increase in the gene product or protein of at least 1.5-fold or at least 2-fold compared to the control. With respect to transcript levels, and unless otherwise defined herein, strong overexpression refers to log2(fold change) > 6. With respect to transcript levels, and unless otherwise defined herein, weak overexpression refers to log2(fold change) < 2. With respect to transcript levels, and unless otherwise defined herein, moderately strong overexpression refers to log2(fold change) ≥ 2 and ≤ 6.

[0034] Expression of a gene product or protein as described herein is "absent or reduced" if the respective gene is deleted or mutated in such a way that the gene product is either not expressed at all or is expressed in a significantly reduced amount (e.g., less than 0.75-fold or less than 0.5-fold). Expression of a gene product or protein as described herein is also considered to be absent or reduced if the gene product or protein has lost functionality, e.g., in a transient or permanent manner (e.g., by mutation or knockdown). Methods for monitoring the amount and / or activity of a gene product or protein are known in the art and are also described herein in an exemplary manner. In general, suitable methods for obtaining absent or reduced expression of a gene product are methods that alter the genetic sequence or element of gene expression (e.g., by deletion or point mutation) and / or methods that negatively affect the transcription and translation of the gene, or the activity or half-life of the gene product (protein).

[0035] Unless otherwise specified, the symbol "Δ" (delta) refers to a "deletion mutant", i.e., a mutant in which a specific gene sequence is at least partially deleted.

[0036] The "early growth phase" is the period when the Actinoplanes strain adapts to the medium and the cell dry weight is less than 3 g·L -1 After adaptation to the environment, the culture metabolizes the nutrients supplied by the medium and begins to grow. Actinoplanes grows within spherical mycelia and can only expand outside the sphere, so the central cells are shielded from nutrients and have only limited space for cell division. Therefore, only the cells in the outer layer of the spherical mycelium are dividing. As a result, the growth of Actinoplanes is linear rather than exponential, in contrast to other bacteria that grow as single cells. The growth phase is called the "linear growth phase" for Actinoplanes species and begins at a cell dry weight of 3 g·L -1 The "stationary phase" is defined as the growth phase when the cells reach the capacity limits (of space and nutrients) respectively and growth decreases due to the formation of inhibitory by-products or other chemical and physical factors such as changes in osmolarity or pH. The stationary phase is a growth phase where the number of dying cells is equal to the number of dividing cells. This phase usually begins at a cell dry weight of 16 - 18 g·L -1 in maltose minimal medium.

[0037] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating the nucleic acid molecule to which it is ligated.

[0038] The term "expression cassette", as used herein, refers to a nucleic acid molecule containing at least one gene and regulatory sequences (e.g., a promoter) for expression.

[0039] A "promoter" is a nucleic acid sequence that directs the initiation of transcription of a specific gene.

[0040] As defined herein, a "strong promoter" is a promoter that results in at least 5·10 -4 [L·g -1 ·min -1 of normalized glucuronidase activity and / or results in a 350-fold relative transcription (log2(fold change)) of the gusA gene compared to a pGUS control vector without a promoter. A detailed description of the method for characterizing promoter strength is provided in the Examples and (Schaffert et al., 2019).

[0041] Examples include the following promoters · apm: 9.2·10 -4 [L·g -1 ·min -1 and log2(fold change) = 360.78 · ermE * : 9.7·10 -4 [L·g -1 ·min -1 and log2(fold change) = 291.03 · katE: 5.1·10 -4 [L·g -1 ·min -1 and log2(fold change) = 342.51 · moeE5: 9.7·10 -4 [L·g -1 ·min -1 and log2(fold change) = 329.32 · gapDH: 11.5·10 -4 [L·g -1 ·min -1 and log2(fold change) = 931.45, and · actP: 22.9·10 -4 [L·g -1 ·min -1 .

[0042] A "moderately strong promoter" is defined as a promoter that results in at least 1·10 -4[L·g -1 ·min -1 results in normalized glucuronidase activity and / or a 10-fold relative transcription (log2(fold change)) of the gusA gene compared to the promoterless pGUS control vector. Examples include the following promoters · efp: 3.1·10-4 [L·g-1·min-1] and log2(fold change) = 53.08 · cdaR: 3.1·10-4 [L·g-1·min-1] and log2(fold change) = 86.82 · rpsL: 3.5·10-4 [L·g-1·min-1] and log2(fold change) = 98.53 · rpsJ: 3.7·10-4 [L·g-1·min-1] and log2(fold change) = 123.97 · cgt: 2.5·10-4 [L·g-1·min-1] and log2(fold change) = 347.29, and · tipA: 4.2·10-4 [L·g-1·min-1] and log2(fold change) = 191.

[0043] In some cases, moderately strong promoters result in at least 1·10 -4 [L·g -1 ·min -1 and 5·10 -4 [L·g -1 ·min -1 of normalized glucuronidase activity.

[0044] A "weak promoter" is defined as a promoter that results in less than 1·10 -4 [L·g -1 ·min -1 of normalized glucuronidase activity and / or a less than 10-fold relative transcription (log2(fold change)) of the gusA gene compared to the promoterless pGUS control vector.

[0045] The term "Cgt" (ACSP50_5024, formerly ACPL_5091) refers to an extracellular small carbohydrate-binding protein and was previously described as cyclomaltodextrin glucanotransferase due to its high similarity to the C-terminal domain of cyclodextrin glycosyltransferase obtained from Actinoplanes sp. (e.g., strain ATCC 31044 / CBS 674.73 / SE50 / 110). The Cgt protein is encoded by the gene cgt. The sequence is described herein (SEQ ID NO: 20) or is accessible via the UniProt identifier G8S155 (G8S155_ACTS5). There may be different isoforms and variants for different strains, and all of these are encompassed by this term. It is clear that sequences with such functionally silent mutations are equivalent with respect to the initial sequence if specific mutations can be exchanged without changing the described catalytic properties of the initial sequence. Furthermore, this protein can undergo various modifications (e.g., synthetic or naturally occurring modifications).

[0046] The term "AcbB" (ACSP50_3608, formerly ACPL_3681) refers to dTDP-D-glucose-4,6-dehydratase obtained from the Actinoplanes sp. strain, such as strain ATCC 31044 / CBS 674.73 / SE50 / 110, which is probably involved in the biosynthesis of the acarbose acarviosyl moiety. The AcbB protein is encoded by the acbB gene. The sequence is described herein (SEQ ID NO: 13) or is accessible via the UniProt identifier Q9ZAE8 (RMLB_ACTS5). There may be different isoforms and variants for different strains, all of which are encompassed by this term. If specific mutations can be exchanged without changing the described catalytic properties of the initial sequence, it is clear that sequences with such functionally silent mutations are equivalent with respect to the initial sequence. Furthermore, this protein may undergo various modifications (e.g., synthetic or naturally occurring modifications).

[0047] The term "GtaB" also "GalU" (ACSP50_7820, formerly ACPL_7811) refers to a UTP-glucose-1-phosphate uridylyltransferase obtained from the Actinoplanes sp., e.g., strain ATCC 31044 / CBS 674.73 / SE50 / 110. GtaB is thought to catalyze the interconversion of glucose-1P and UDP-glucose and to be involved in the precursor supply of acarbose. The GtaB protein is encoded by the gtaB gene. The sequence is described herein (SEQ ID NO: 19) or is accessible via the UniProt identifier G8S608 (ACPL_7811). There may be different isoforms and variants for different strains, all of which are encompassed by this term. It is clear that sequences with such functionally silent mutations are equivalent with respect to the initial sequence if specific mutations can be exchanged without changing the described catalytic properties of the initial sequence. Furthermore, this protein can undergo various modifications (e.g., synthetic or naturally occurring modifications).

[0048] As defined herein, a "gene essential for carotenoid synthesis" is defined as a gene that is positively required for the synthesis of carotenoids. Actinoplanes is known to produce various soluble pigments, including the yellow, orange, and pink pigments of carotenoids. In Actinoplanes, the set of genes essential for carotenoid synthesis includes genes from the MEP / DOXP pathway, genes of terpene cluster 1, genes of terpene cluster 2a, genes of terpene cluster 2b, and genes of the camphene-like monoterpene biosynthetic terpene cluster 3. The genes of the MEP / DOXP pathway include the following i. The 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID NO: 23), ii. The 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID NO: 24), iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID NO: 25), iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID NO: 26), v. 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID NO: 27), vi. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene ispF, ACSP50_8046, SEQ ID NO: 28), and / or vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID NO: 29).

[0049] The genes of terpene cluster 1 include the following i. Isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID NO: 30), ii. Zeta-carotene desaturase gene crtI (ACSP50_0147, SEQ ID NO: 10), iii. Polyprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID NO: 31), iv Phytoene synthase gene crtB (ACSP50_0149, SEQ ID NO: 32), v. Deoxyribodipyrimidine photolyase gene (ACSP50_0150, SEQ ID NO: 33), or vi. Pyridine nucleotide-disulfide oxidoreductase gene (ACSP50_0151, SEQ ID NO: 34).

[0050] The genes of terpene cluster 2a include the following i. Transcription regulator gene (ACSP50_1631, SEQ ID NO: 35), ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID NO: 36), iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID NO: 37), iv. Polyprenyl synthetase (farnesyl pyrophosphate synthetase 2 gene fps2 / crtE (ACSP50_1634, SEQ ID NO: 38), and v. Methylene tetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID NO: 39).

[0051] The genes of terpene cluster 2b include the following i. LysR-family transcriptional regulator gene (ACSP50_1650, SEQ ID NO: 40), ii. Methyltransferase type 11 gene (ACSP50_1651, SEQ ID NO: 41), iii. CDP-alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID NO: 42), iv. Zeta-carotene desaturase (crtI-family) gene crtD (ACSP50_1653, SEQ ID NO: 43), v. Glycosyltransferase gene cruC (ACSP50_1654, SEQ ID NO: 44), vi. Hypothetical protein (put.membrane prot,) gene cruF, (ACSP50_1655, SEQ ID NO: 45), vii. GCN5-family acetyltransferase gene (ACSP50_1656, SEQ ID NO: 46), viii. Monooxygenase gene (ACSP50_1657, SEQ ID NO: 47), and ix. Short-chain dehydrogenase gene (ACSP50_1658, SEQ ID NO: 48).

[0052] Another gene essential for carotenoid synthesis is the polyprenyl synthetase gene crtE (ACSP50_3873, SEQ ID NO: 49).

[0053] The genes of the camphor-like monoterpene biosynthesis terpene cluster 3 include the following i. Transcription regulatory factor (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID NO: 104), ii. Camphor synthase gene (ACSP50_1950, SEQ ID NO: 50), iii. Methyltransferase (SAM-dependent) type 11 gene (ACSP50_1951, SEQ ID NO: 105), iv. Glycosyl-hydrolase gene (ACSP50_1952, SEQ ID NO: 106), and v. Oxidoreductase / ald / keto reductase (ACSP50_1953, SEQ ID NO: 107).

[0054] Embodiments The actinomycete (Actinomycetales) strain Actinoplanes sp. SE50 / 110 was used as the model strain of the present invention, but it is obvious to those skilled in the art that the general mechanisms and discoveries can also be applied to other acarbose-producing strains such as the strains currently used for the commercial production of acarbose. According to some embodiments, the actinomycete strain is a Micromonosporaceae strain. According to some embodiments, the actinomycete strain is an Actinoplanes strain. According to some embodiments, the actinomycete 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 therefrom. In some embodiments, the actinomycete strain is an Actinoplanes strain commercially used for acarbose production. In some embodiments, the actinomycete strain is, for example, an Actinoplanes strain 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 in EP2601209B1 and CN103298828B, or a strain derived therefrom.

[0055] Improvements in acarbose production refer to an increase in the yield of acarbose (either overall or relative to cell growth) over a specific period of time and / or an improvement in the purity of acarbose, e.g., a decrease in by-products such as component C and / or acarbose analogues. Cultivation of Actinoplanes strains can occur as known in the art or as described herein. In some embodiments, the cultivation of Actinoplanes strains is carried out in maltose minimal medium.

[0056] According to a first aspect of the present invention, there is provided a method of manipulating an actinomycete strain, such as an Actinoplanes strain, for improved production of acarbose.

[0057] According to some first embodiments according to the first aspect, the method according to the first aspect comprises manipulating the actinomycete strain for the loss or attenuation of the expression of the extracellular small carbohydrate-binding protein Cgt (SEQ ID NO: 20).

[0058] Surprisingly, the loss of the carbohydrate-binding protein Cgt (SEQ ID NO: 20) resulted in an improvement in acarbose production. An increase in the final acarbose yield of 8.3 - 16.6% was achieved in three independent shake flask cultures (see Example "Δcgt shows improved acarbose formation in maltose minimal medium", Figures 18, 19, Table E10, Table E11).

[0059] Furthermore, compared with the wild type, the gene deletion mutant Δcgt did not show an obvious growth phenotype in screening experiments testing different carbon sources, or under carbon limitation conditions (see Examples "Analysis of cgt expression during growth on different carbon sources", "Analysis of cgt expression on different carbon sources or under carbon limitation conditions", Figures 12, 13, 14), or in screening experiments under pH stress and osmolyte stress (see Examples "cgt does not affect osmotic or pH tolerance", Figures 15, 16, 17). The inventors were further able to show that the deletion of cgt did not negatively affect the expression of the acarbose biosynthetic genes (see Example "Δcgt does not affect the expression of acarbose biosynthetic genes", Figure 20).

[0060] 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 transported to the extracellular space, which accounts for approximately 8% of the entire secreted proteome. The inventors analyzed the distribution of CBM-20 single-domain proteins in the prokaryotic world by BlastP analysis. Interestingly, specific CBM-20 domain proteins were found in only 17 other species (see 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, the deletion or reduced expression of cgt relaxes energy and resources such as ATP and amino acids. These resources can then be redirected towards the biosynthesis of acarbose, a growth-related product.

[0061] According to some embodiments of the first aspect, the method involves a deletion or mutation of the gene encoding the extracellular small carbohydrate-binding protein Cgt (SEQ ID NO: 20). The establishment of the intergeneric conjugation system (Gren et al., 2016) and the CRISPR / Cas9 technology (Wolf et al., 2016) enables genome editing in the actinomycetes species SE50 / 110. In some embodiments of the first aspect, the manipulation of the actinomycetes strain for the loss or attenuation of expression can be carried out using the CRISPR / Cas9 technology. In some embodiments, the manipulation of the actinomycetes strain for the loss or attenuation of expression can occur as described by (Wolf et al., 2016). In some embodiments, the manipulation of the actinomycetes strain for the loss or attenuation of expression can occur as described herein, for example, in the examples "Deletion of gene cgt by CRISPR / Cas9 technology" or "Deletion system based on homologous recombination with cytosine deaminase CodA and counterselection".

[0062] For example, the inventors have successfully established a novel deletion system by homologous recombination, which uses an integrase-free vector backbone and CodA for counterselection, as described by Zhao et al. (2017).

[0063] According to some second embodiments of the first aspect, the method according to the first aspect involves manipulating an actinomycetes strain for the loss or attenuation of the expression of at least one gene essential for carotenoid synthesis. In some embodiments, the carotenoid is the orange pigment of actinomycetes or its derivatives. In some different or same embodiments, the carotenoid is a C40-carotenoid.

[0064] The manipulation of the actinomycetes strain for the loss or attenuation of expression can occur as previously described for this aspect. According to some embodiments of the first aspect, the method involves a deletion or mutation of a gene essential for carotenoid synthesis.

[0065] Actinoplanes is known to produce various soluble pigments including yellow, orange, and pink pigments of carotenoids (Parenti and Coronelli, 1979). The inventors observed that strong pigment deposition was associated with a loss of acarbose production. This was confirmed by comparing the growth and acarbose yield of cultures exposed to light with those covered from light (see Example "Light-dependent carotenoid formation and oxidative stress decrease acarbose production in Actinoplanes sp. SE50 / 110", Figure 22). Although carotenoid formation was induced, the acarbose production and growth of Actinoplanes sp. SE50 / 110 strongly decreased when exposed to bulb light (Figure 22). Overall, a loss of 39% of the final acarbose concentration was monitored.

[0066] From these findings, it is reasonable that not only are the produced pigments not essential (e.g., in a technical setting for commercial acarbose production), but that acarbose formation can be improved by taking advantage of decreasing or depleting carotenoid synthesis in Actinoplanes. For this purpose, a method according to a first aspect involves decreasing or depleting the expression of at least one gene essential for carotenoid synthesis.

[0067] The inventors were able to further reconstruct carotenoid production in Actinoplanes sp. SE50 / 110 (see Example "Analysis of the functional relevance of carotenoid formation", Figure 21). The set of genes essential for carotenoid synthesis in Actinoplanes includes genes from the MEP / DOXP pathway, genes of terpene cluster 1, genes of terpene cluster 2a, genes of terpene cluster 2b, and genes of the camphene-like monoterpene biosynthesis terpene cluster 3.

[0068] According to some embodiments of this aspect and embodiment, at least one gene essential for carotenoid synthesis is a gene of the MEP / DOXP pathway as follows i. 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID NO: 23), ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID NO: 24), iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID NO: 25), iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID NO: 26), v. 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID NO: 27), vi. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene ispF, ACSP50_8046, SEQ ID NO: 28), and / or vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID NO: 29).

[0069] According to some embodiments of this aspect and embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 1 as follows i. Isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID NO: 30), ii. Zeta-carotene desaturase gene crtI (ACSP50_0147, SEQ ID NO: 10), iii. Polyprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID NO: 31), iv. Phytoene synthase gene crtB (ACSP50_0149, SEQ ID NO: 32), v. Deoxyribodipyrimidine photolyase gene (ACSP50_0150, SEQ ID NO: 33), or vi. Pyridine nucleotide-disulfide oxidoreductase gene (ACSP50_0151, SEQ ID NO: 34).

[0070] According to some embodiments of this aspect and the embodiments, at least one gene essential for carotenoid synthesis is the zeta-carotene desaturase gene crtI (ACSP50_0147, SEQ ID NO: 10). As described above, carotenoid formation is not required under laboratory conditions. To improve acarbose production, especially by deleting the central gene crtI, switching off the concurrent carotenoid biosynthesis pathway can be used in strain development.

[0071] According to some embodiments of this aspect and the embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2a as follows i. Transcription regulator gene (ACSP50_1631, SEQ ID NO: 35), ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID NO: 36), iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID NO: 37), iv. Polyprenyl synthetase (farnesyl pyrophosphate synthetase 2 gene fps2 / crtE (ACSP50_1634, SEQ ID NO: 38), or v. Methylene tetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID NO: 39).

[0072] According to some embodiments of the current aspect and the embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2b as follows i. LysR-family transcription regulator gene (ACSP50_1650, SEQ ID NO: 40), ii. Methyltransferase type 11 gene (ACSP50_1651, SEQ ID NO: 41), iii. CDP - alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID NO: 42), iv. Zeta - phytoene desaturase (crtI - family) gene crtD (ACSP50_1653, SEQ ID NO: 43), v. Glycosyltransferase gene cruC (ACSP50_1654, SEQ ID NO: 44), vi. Hypothetical protein (put.membrane prot,) gene cruF, (ACSP50_1655, SEQ ID NO: 45), vii. GCN5 family acetyltransferase gene (ACSP50_1656, SEQ ID NO: 46), viii. Monooxygenase gene (ACSP50_1657, SEQ ID NO: 47), or ix. Short - chain dehydrogenase gene (ACSP50_1658, SEQ ID NO: 48).

[0073] According to some embodiments of this aspect and the embodiments, at least one gene essential for carotenoid synthesis is the polyprenyl synthase gene crtE (ACSP50_3873, SEQ ID NO: 49).

[0074] According to some embodiments of this aspect and the embodiments, at least one gene essential for carotenoid synthesis is a gene of the following camphene - like monoterpene biosynthesis terpene cluster 3 i. Transcription regulator (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID NO: 104), ii. Camphene synthase gene (ACSP50_1950, SEQ ID NO: 50), iii. Methyltransferase (SAM - dependent) type 11 gene (ACSP50_1951, SEQ ID NO: 105), iv. Glycosyl - hydrolase gene (ACSP50_1952, SEQ ID NO: 106), or v. Oxidoreductase / ald / keto reductase (ACSP50_1953, SEQ ID NO: 107).

[0075] Since carotenoids affect membrane fluidity, the absence of carotenoids, particularly C40-carotenoids, can also affect the surface and hyphal structure of the Actinoplanes species SE50 / 110. With respect to production, the breakdown of mycelial clumps is advantageous for increasing the mycelial surface and the number of biochemically available cells.

[0076] According to some further embodiments, the method according to the first aspect comprises engineering an actinomycete strain for overexpression of the MerR- / HTH transcriptional regulator gene merR (ACSP50_0145, SEQ ID NO: 11). Engineering of the actinomycete strain for overexpression can occur as described elsewhere herein.

[0077] In addition to the above genes essential for carotenoid synthesis, the inventors surprisingly identified a transcriptional repressor for carotenoid synthesis among the genes of terpene cluster 1: ACSP50_0145 (SEQ ID NO: 11, MerR- / HTH transcriptional regulator gene merR) (see Example "Deletion of merR in SE50 / 110 induces carotenoid formation without exposure to light", Figure 24). By CRISPR / Cas9 deletion of the corresponding gene in SE50 / 110, carotenoid formation was strongly induced without exposure to light (Figures 24B and C). Consistent with this, a decrease in acarbose production was found. When irradiated, both the wild type and ΔmerR were strongly pigmented and the final acarbose concentration was similar for both strains, reaching about 0.52 g·L -1 and reached (Figures 24B and D). This corresponds to a decrease in acarbose formation of about 38% compared to the wild type under dark conditions (reaching 0.83 g·L -1 This is consistent with previous growth experiments of the wild type. Under dark conditions, ΔmerR was lower than the wild type (0.70 g·L -1Produce less than about 15% of acarbose (Figure 24D). Without being bound by theory, these production losses are thought to be caused by waste of resources due to carotenoid formation in the deletion mutants (Figure 24C). In conclusion, the production losses (38 - 39%) under light conditions may be attributed to further light-induced stress in both the deletion mutants and the wild type.

[0078] According to some third embodiments according to the first aspect, the method comprises engineering a Streptomyces strain for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13).

[0079] According to the present invention, surprisingly, overexpression of the acb gene encoding dTDP-D-glucose-4,6-dehydratase AcbB was found to significantly increase the final acarbose concentration by about 50%. This was particularly surprising since other genes of the Acb cluster, such as AcbC, did not result in an improvement in acarbose formation. Furthermore, the increase observed was superior compared to the increase observed for overexpression of the complete Acb cluster as described by Zhao et al. (Zhao, Xie et al., 2017).

[0080] According to some embodiments, the strain does not comprise engineering the Streptomyces strain for overexpression of other genes of the Acb cluster except AcbA.

[0081] dTDP-D-glucose-4,6-dehydratase AcbB appears to be involved in the production of activated amino sugars from D-glucose-1P, which is a supply route for acarbose biosynthesis (Figure 1): Without being bound by theory, increased AcbB activity was surprisingly also found to improve the supply of modified precursors.

[0082] The overexpression of AcbB described herein refers to an increase in the expression of AcbB compared to the wild type or a specific reference strain / control. For example, the overexpression of the gene product can be an increase during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time.

[0083] Preferably, as described herein, the overexpression of AcbB refers to an increase in AcbB transcript and / or protein by a factor of at least 1.5 or at least 2 compared to the control. With respect to the amount of AcbB transcript, and unless otherwise defined herein, strong overexpression refers to log2(fold change) > 6. With respect to the amount of AcbB transcript, unless otherwise defined herein, moderately strong overexpression refers to log2(fold change) ≥ 2 and ≤ 6.

[0084] According to some embodiments, the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein by a factor of at least 1.5 or at least 2 log2(fold change) during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time.

[0085] According to some embodiments, the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein with log2(fold change) ≥ 2 and ≤ 6 during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time such as during the initial growth phase and / or during the linear growth phase.

[0086] According to some embodiments, the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein with log2(fold change) > 3 and < 5 during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time.

[0087] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcripts and / or proteins with log2(fold change)>6 during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase during any other time.

[0088] In overexpression mutants with expression vectors containing heterologous promoters, the relative transcription of acbB decelerated from 4.06-fold to 3.33-fold (log2(fold change)) during two sampling times in pSETT4tip::acbB (moderately strong promoter) and from 6.54 to 2.05-fold in pSETT4gap::acbB (strong promoter) (see the example "Moderate overexpression of acbB leads to improved acarbose formation").

[0089] According to some embodiments, the engineering of actinomycete strains for overexpression of genes according to the first aspect can occur by any method known in the art or described herein.

[0090] As described in the example "Moderate overexpression of acbB leads to improvement of acarbose formation", two pSETT4-based overexpression mutants were generated in which acbB was transcribed under the control of a moderately strong tipA-promoter or a strong gapDH-promoter. The native promoter was used as a control in both the pSET152- and pSETT4-vector backgrounds. In particular, mutants with acbB transcribed under the control of the heterologous tipA-promoter enhanced acarbose production compared to the control strain (Figures 27, 28). The yield coefficients increased by 48.6 and 51.9% compared to the empty vector control. The use of the strong gapDH promoter was found to slightly increase the acarbose yield coefficient (Figure 28).

[0091] According to some embodiments, the manipulation of the actinomycete strain for overexpression of a gene according to the first aspect can be performed by introducing into the actinomycete strain a vector containing an expression cassette of AcbB (SEQ ID NO: 13). In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. The vector is derived from another vector when it contains at least one, two, three, four elements of the second vector.

[0092] According to some embodiments, the manipulation of the actinomycete strain for overexpression of a gene according to the first aspect can be performed by introducing into the actinomycete strain a vector containing an expression cassette of AcbB (SEQ ID NO: 13). In some of these embodiments or some of other embodiments, the expression cassette is under the control of a moderately strong promoter and has a normalized glucuronidase activity of at least 1x10 -4 , preferably 1x10 -4 ~5×10 -4 [L·g -1 ·min -1 in the glucuronidase assay, as described elsewhere herein, for example. In some embodiments, the promoter is selected from the efp promoter (SEQ ID NO: 92), the cdaR promoter (SEQ ID NO: 97), the rpsL promoter (SEQ ID NO: 99), the rpsJ promoter (SEQ ID NO: 93), the cgt promoter (SEQ ID NO: 91), or the 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 using pSETT4tip::acbB. See FIGS. 27 and 28.

[0093] In some aspects, the expression cassette is under the control of a strong promoter and has a normalized glucuronidase activity of at least 5×10 -5 [L·g -1 ·min -1It is characterized by the normalized glucuronidase activity. In some embodiments, the promoter is the apm promoter (SEQ ID NO: 96), ermE * The promoter (SEQ ID NO: 98), katE promoter (SEQ ID NO: 94), moeE5 promoter (SEQ ID NO: 95) or gapDH promoter (SEQ ID NO: 82) is selected from.

[0094] According to some embodiments, the method according to the first aspect involves manipulating a streptomyces strain for moderate overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) and optionally AcbA (SEQ ID NO: 12). In some embodiments, it is compatible with all other embodiments described herein unless otherwise specified, and the genetic manipulation does not result in an increase in transcripts and / or proteins for Acb genes other than AcbB and AcbA with log2 (fold change) ≥ 2. In some embodiments that are also compatible with all other embodiments described herein, the genetic manipulation does not result in an increase in transcripts and / or proteins for AcbC with log2 (fold change) ≥ 2.

[0095] Due to the overexpression of AcbB, further genes of the acb gene cluster were not significantly affected, as shown for acbA and acbV, for example, in the early growth phase (Figure 30). The only exception is the slightly higher transcript level of acbA in pSETT4tip::acbB (log2 (fold change) = 1.87).

[0096] According to some embodiments, the method according to the first aspect involves manipulating a streptomyces strain for overexpression of AcbB (SEQ ID NO: 13) and AcbS (ACSP50_3596) and / or AcbI (ACSP50_3599).

[0097] Overexpression of AcbS and / or AcbI (additional) can enhance the transfer reaction of amino sugars to cyclitol precursors. According to this model (see Figure 1), this reaction is catalyzed by AcbS (ACSP50_3596) or AcbI (ACSP50_3599).

[0098] According to some embodiments, the method according to the first aspect involves engineering a streptomycete 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 AcbI (ACSP50_3599)). Without being bound by theory, this combination may be able to enhance both acarbose synthesis strands.

[0099] According to some fourth embodiments according to the first aspect, the method involves engineering a streptomycete strain for overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID NO: 19).

[0100] A 8.5% increase in the final acarbose concentration was observed with moderate overexpression of gtaB (see Examples "Moderate overexpression of gtaB leads to improved acarbose formation", Figures 32 and 33). Interestingly, acarbose formation increases particularly from the late logarithmic growth phase to the stationary growth phase (Figure 32). Without being bound by theory, this may result from improved deployment of the precursor glucose-1P (see Figure 34).

[0101] Overexpression of GtaB (SEQ ID NO: 19) as described herein refers to an increase in the expression of GtaB transcript and / or protein compared to wild type or a specific reference strain / control. For example, overexpression of the gene product can be an increase during the early growth phase and / or the logarithmic growth phase and / or the stationary phase, and / or an increase at any other time.

[0102] Preferably, overexpression is an increase in GtaB transcript and / or protein by a factor of at least 1.5 or at least 2 compared to a control. For GtaB transcript levels, and unless otherwise defined herein, strong overexpression refers to log2(fold change)>6. For GtaB transcript levels, and unless otherwise defined herein, moderately strong overexpression refers to log2(fold change)≥2 and ≤6.

[0103] According to some embodiments, overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in GtaB expression by a factor of at least 1.5 or at least 2 log2(fold change) during at least 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.

[0104] In one of the overexpression mutants described herein, the relative transcript level of the gene gtaB is increased 2.64-fold (log2(fold change)) (Figure 33).

[0105] According to some embodiments, overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in GtaB transcript and / or protein expression by log2(fold change)≥2 and ≤6 during at least 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, overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in GtaB expression by log2(fold change)≥3 and ≤5 during at least 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.

[0106] According to some embodiments, overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in GtaB expression by log2(fold change)≥6 during at least the early growth phase and / or during the linear growth phase and / or during the stationary phase.

[0107] According to some embodiments, the engineering of the actinomycete strain for overexpression of a gene according to the first aspect can be performed by introducing into the actinomycete strain a vector comprising an expression cassette of GtaB (SEQ ID NO: 19). In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. The vector is derived from another vector if it contains at least one, two, three, four elements of the second vector.

[0108] According to some embodiments, the engineering of the actinomycete strain for overexpression of a gene according to the first aspect can be performed by introducing into the actinomycete strain a vector comprising an expression cassette of GtaB (SEQ ID NO: 19).

[0109] In some of these or other embodiments, the expression cassette is under the control of a moderately strong promoter and has, for example, in a glucuronidase assay, as described elsewhere herein, a normalized glucuronidase activity of 1×10 -4 ~5×10 -5 [L·g -1 ·min -1 . In some embodiments, the promoter is selected from the efp promoter (SEQ ID NO: 92), the cdaR promoter (SEQ ID NO: 97), the rpsL promoter (SEQ ID NO: 99), the rpsJ promoter (SEQ ID NO: 93), the cgt promoter (SEQ ID NO: 91), or the 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, using pSETT4tip::gtaB. See FIGS. 32 and 33.

[0110] In some aspects, the expression cassette is under the control of a strong promoter and has, for example, in a glucuronidase assay, as described elsewhere herein, a normalized glucuronidase activity of at least 5×10 -5 [L·g -1 ·min -1It is characterized by the normalized glucuronidase activity. In some embodiments, the promoter is the apm promoter (SEQ ID NO: 96), ermE * The promoter (SEQ ID NO: 98), katE promoter (SEQ ID NO: 94), moeE5 promoter (SEQ ID NO: 95) or gapDH promoter (SEQ ID NO: 82) is selected from.

[0111] According to some further embodiments or the same embodiment of the first aspect, the method comprises engineering an actinomycete strain for moderate overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) and GtaB (SEQ ID NO: 19).

[0112] It was surprising that overexpression of GtaB induced improved acarbose formation. A positive effect on acarbose production was observed by moderate overexpression of acbB (e.g., by use of the tipA-promoter), producing about 50% or more acarbose in two independent cultures. Thus, improvement of acarbose biosynthesis by overexpression of the specific acb gene AcbB was achieved. Furthermore, a moderate overexpression of gtaB resulted in an 8.5% increase in the final acarbose concentration. The combined overexpression of acbB and gtaB is thought to improve the metabolic flux through amino sugar biosynthesis and further enhance acarbose production.

[0113] Without being bound by theory, strong overexpression of AcbB induced only a slight increase in acarbose production compared to moderate strong overexpression of AcbB. This is thought to be due to an imbalance in glucose-phosphate metabolism that occurs upon high overexpression of AcbB. Overexpression of gtaB may cure this imbalance, and the combined overexpression of both acbB and gtaB is thought to lead to a further increase in acarbose production.

[0114] Interestingly, a significant decrease in mass M / z = 545 [M-H+] was found in pSETT4tip::gtaB (a decrease of about 48%), which may correspond to dTDP-4-keto-6-deoxy-D-glucose, the proposed product of AcbB. This may indicate that the metabolic flux through the synthetic pathway is more balanced as the accumulation of this metabolite is decreased compared to the empty vector control and the AcbB-overexpressing mutant (Figure 34).

[0115] According to some embodiments, the method according to the first aspect comprises (i) for the disappearance or attenuated expression of the extracellular small carbohydrate-binding protein Cgt (SEQ ID NO: 20), and / or (ii) for the disappearance or attenuated expression of at least one gene involved in carotenoid synthesis, and / or (iii) for the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13), and / or (iv) for the overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID NO: 19), manipulating the actinomycete strain.

[0116] According to some embodiments, the method according to the first aspect further comprises manipulating the actinomycete strain for the disappearance or attenuated expression of treY.

[0117] According to some embodiments, the method according to the first aspect further comprises (i) deletion or mutation of the gene encoding the extracellular small carbohydrate-binding protein Cgt (SEQ ID NO: 20), and / or, (ii) deletion or mutation of at least one gene involved in carotenoid synthesis, and / or, (iii) introducing a vector containing an expression cassette for AcbB (SEQ ID NO: 13) into the actinomycete strain, and / or, (iv) introducing a vector containing an expression cassette for GtaB (SEQ ID NO: 19) into the actinomycete strain, comprises

[0118] According to some embodiments, the expression cassette according to (iii) and / or (iv) is under the control of a moderately strong promoter and, in a glucuronidase assay, 1x10 -4 ~5×10 -5 [L·g -1 ·min -1 of normalized glucuronidase activity.

[0119] According to a second aspect, there is provided a streptomycete strain, such as an Actinoplanes strain, for the production of acarbose. According to some embodiments, the streptomycete strain is a strain generated by the method according to the first aspect. According to some other embodiments, the streptomycete strain is genetically engineered for the loss or attenuation of the expression of the extracellular small carbohydrate-binding protein Cgt (SEQ ID NO: 20). According to some embodiments, the streptomycete strain is a Δcgt mutant. The Δcgt mutant is a mutant of a streptomycete strain in which the gene Cgt (SEQ ID NO: 20) is at least partially deleted or inverted.

[0120] According to some of these or other embodiments, the streptomycete strain is genetically engineered for the loss or attenuation of the expression of at least one gene essential for carotenoid synthesis. According to some embodiments, at least one gene essential for carotenoid synthesis is at least partially deleted or inverted. According to some of these embodiments, at least one gene essential for carotenoid synthesis comprises at least one gene selected from any of the following a. Genes of the MEP / DOXP pathway, such as i 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID NO: 23), ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID NO: 24), iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID NO: 25), iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID NO: 26), v. 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID NO: 27), vi. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene ispF, ACSP50_8046, SEQ ID NO: 28), and / or vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID NO: 29), b. Genes of terpene cluster 1, for example, i. Isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID NO: 30), ii. Zeta-carotene desaturase gene crtI (ACSP50_0147, SEQ ID NO: 10), iii. Polyprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID NO: 31), iv. Phytoene synthase gene crtB (ACSP50_0149, SEQ ID NO: 32), v. Deoxyribodipyrimidine photolyase gene (ACSP50_0150, SEQ ID NO: 33), or vi. Pyridine nucleotide-disulfide oxidoreductase gene (ACSP50_0151, SEQ ID NO: 34), c. Genes of terpene cluster 2a, for example, i. Transcription regulator gene (ACSP50_1631, SEQ ID NO: 35), ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID NO: 36), iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID NO: 37), iv. Polyprenyl synthetase (farnesyl pyrophosphate synthetase 2 gene fps2 / crtE (ACSP50_1634, SEQ ID NO: 38), or v. Methylene tetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID NO: 39), d. Genes of terpene cluster 2b, for example, i. LysR-family transcriptional regulator gene (ACSP50_1650, SEQ ID NO: 40), ii. Methyltransferase type 11 gene (ACSP50_1651, SEQ ID NO: 41), iii. CDP-alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID NO: 42), iv. Zeta-carotene desaturase (crtI-family) gene crtD (ACSP50_1653, SEQ ID NO: 43), v. Glycosyltransferase gene cruC (ACSP50_1654, SEQ ID NO: 44), vi. Hypothetical protein (put.membrane prot,) gene cruF, (ACSP50_1655, SEQ ID NO: 45), vii. GCN5-family acetyltransferase gene (ACSP50_1656, SEQ ID NO: 46), viii. Monooxygenase gene (ACSP50_1657, SEQ ID NO: 47), ix. Short-chain dehydrogenase gene (ACSP50_1658, SEQ ID NO: 48), e. Polyprenyl synthetase gene crtE (ACSP50_3873, SEQ ID NO: 49), or f. Genes of the camphene-like monoterpene biosynthetic terpene cluster 3, for example, i. Transcriptional regulator (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID NO: 104), ii. Camphene synthase gene (ACSP50_1950, SEQ ID NO: 50), iii. Methyltransferase (SAM-dependent) type 11 gene (ACSP50_1951, SEQ ID NO: 105), iv. Glycosyl-hydrolase gene (ACSP50_1952, SEQ ID NO: 106), v. Oxidoreductase / ald / keto reductase (ACSP50_1953, SEQ ID NO: 107).

[0121] According to some of these or other embodiments, the actinomycete strain is genetically engineered for overexpression of the MerR- / HTH transcriptional regulator gene merR (ACSP50_0145, SEQ ID NO: 11).

[0122] According to some of these or other embodiments, the actinomycete strain is genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13).

[0123] As described elsewhere herein, overexpression of AcbB refers to an increase in AcbB by a factor of at least 1.5 or at least 2 compared to a control. Preferably, the control is a strain that has not been engineered for specific overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13). For example, the control does not contain a vector containing an expression cassette for AcbB.

[0124] For example, overexpression of a gene product can be an increase during the initial growth phase, during the linear growth phase, during the stationary phase, or during any other time.

[0125] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcripts and / or proteins by a factor of at least 1.5 or at least 2 log2 (fold change) during the initial growth phase, during the linear growth phase, during the stationary phase, or during any other time.

[0126] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein during the initial growth phase, during the linear growth phase, during the stationary phase, with log2 (fold change) ≥ 2 and ≤ 6, or an increase during any other time, such as during the initial growth phase and / or during the linear growth phase.

[0127] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein during the initial growth phase, during the linear growth phase, during the stationary phase, with log2 (fold change) > 3 and < 5, or an increase during any other time.

[0128] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) is an increase in the expression of AcbB transcript and / or protein during the initial growth phase, during the linear growth phase, during the stationary phase, with log2 (fold change) > 6, or an increase during any other time.

[0129] According to some embodiments, a genetically engineered actinomycete strain for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) contains a vector for overexpression of AcbB. According to some of these embodiments, the vector is the vector described herein, preferably according to the embodiments described herein.

[0130] According to some embodiments, a genetically engineered actinomycete strain for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) contains an expression cassette for AcbB (SEQ ID NO: 13) under the control of a moderately strong promoter.

[0131] According to some embodiments, the actinomycete strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO: 13) contains an expression cassette for AcbB (SEQ ID NO: 13) under the control of a strong promoter. Preferably, the promoter is not the native promoter of AcbB.

[0132] According to some of these or other embodiments, the actinomycete strain is genetically engineered for overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID NO: 19).

[0133] Overexpression of GtaB (SEQ ID NO: 19) as described elsewhere herein refers to an increase in the expression of GtaB compared to wild type or a particular reference strain / control. Preferably, the control is a strain that has not been engineered for specific overexpression of GtaB (SEQ ID NO: 19). For example, the control does not contain a vector containing an expression cassette for GtaB (SEQ ID NO: 19). For example, overexpression of the gene product can be an increase during the initial growth phase and / or during the linear growth phase and / or during the stationary phase, and / or an increase at any other time.

[0134] According to some embodiments, overexpression of GtaB is an increase in the expression of GtaB transcripts and / or proteins by a factor of at least 1.5, or at least 2 log2 (fold change) during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time.

[0135] According to some embodiments, overexpression of GtaB is an increase in the expression of GtaB transcripts and / or proteins with log2 (fold change) ≧ 2 and ≦ 6 during the initial growth phase, during the linear growth phase, during the stationary phase, or an increase at any other time such as during the initial growth phase and / or during the linear growth phase.

[0136] According to some embodiments, overexpression of GtaB is an increase in the expression of GtaB transcript and / or protein during the early growth phase, during the linear growth phase, during the stationary phase, with log2(fold change)>3 and <5, or an increase during any other time.

[0137] According to some embodiments, overexpression of GtaB is an increase in the expression of GtaB transcript and / or protein during the early growth phase, during the linear growth phase, during the stationary phase, with log2(fold change)>6, or an increase during any other time.

[0138] According to some embodiments, the actinomycete strain genetically engineered for overexpression of GtaB contains a vector for overexpression of GtaB. According to some of these embodiments, the vector is the vector described herein, preferably according to the embodiments described herein.

[0139] According to some embodiments, the actinomycete strain genetically engineered for overexpression of GtaB (SEQ ID NO: 19) contains an expression cassette for GtaB (SEQ ID NO: 19) under the control of a moderately strong promoter.

[0140] According to some embodiments, the actinomycete strain genetically engineered for overexpression of GtaB (SEQ ID NO: 19) contains an expression cassette for GtaB (SEQ ID NO: 19) under the control of a strong promoter. Preferably, the promoter is not the native promoter of GtaB.

[0141] According to a third aspect, an actinomycete strain, such as an Actinoplanes strain, is provided for use in the production of acarbose.

[0142] According to some embodiments, a method for producing acarbose is provided, the method comprising using an actinomycete strain according to the second aspect.

[0143] For genetic manipulation of Actinoplanes, an expression system is required for overexpression of one or more genes. According to a fourth aspect, an expression vector for Actinoplanes is provided.

[0144] According to some embodiments, the vector according to the fourth aspect comprises a moderately strong promoter characterized by a normalized glucuronidase activity of at least 1x10-4 [L·g-1·min-1] in the glucuronidase assay. In some embodiments, the moderately strong promoter is selected from efp of SEQ ID NO: 92, cdaR of SEQ ID NO: 97, rpsL of SEQ ID NO: 99, rpsJ of SEQ ID NO: 93, cgt of SEQ ID NO: 91, or tipA of SEQ ID NO: 81.

[0145] According to some embodiments, the vector according to the fourth aspect comprises a strong promoter characterized by a normalized glucuronidase activity of at least 5x10-4 [L·g-1·min-1] in the glucuronidase assay. In some embodiments, the strong promoter is selected from apm of SEQ ID NO: 96, ermE* of SEQ ID NO: 98, katE of SEQ ID NO: 94, moeE5 of SEQ ID NO: 95, or gapDH of SEQ ID NO: 82.

[0146] To find further suitable promoters enabling moderate to strong gene expression, promoter screening can be carried out by use of the screening systems of Horbal et al. (2013) and Myronovskyi et al. (2011), which are based on the reporter GusA cloned in the pSET152 vector system (see Figure 3, Table 1).

[0147] In some embodiments, the vector according to the first aspect comprises an expression cassette. Preferably, the vector comprises an expression cassette of AcbB (SEQ ID NO: 13) and / or an expression cassette of GtaB (SEQ ID NO: 19) and / or an expression cassette of MerR.

[0148] In some embodiments, the expression cassette may further comprise a lacZα-gene under the control of a lac-promoter. The lacZα-gene encodes the catalytic domain of β-galactosidase and allows for the rapid selection of integration of target sequences by blue-white selection in the cloning strain Escherichia coli DH5αMCR (NC_017638.1) (Grant et al. 1990).

[0149] Without being bound by theory, the vector according to this aspect contains elements for vector replication, transmission, maintenance, and selection. In some embodiments, at least one of these elements is derived from pSET152.

[0150] In some embodiments, the vector according to this aspect contains a part of the sequence of the pSET152 vector of Bierman et al. (1992).

[0151] Preferably, the vector does not contain the putative antisense promoter according to SEQ ID NO: 108 and / or SEQ ID NO: 109. These antisense promoters were identified by the inventors by sequencing of the 5'-primary transcript library and impair the compatibility of the vector pSET152. Briefly, the identification was performed by sequencing of an enriched primary transcript library. Two putative promoters were identified behind the target gene in the antisense orientation (Figure 26). These two pseudo-promoters were removed to prevent antisense transcription.

[0152] Furthermore, a T4-terminator was introduced in the opposite direction behind the expression cassette to prevent further putative antisense readthrough (see, for example, FIG. 6). In some embodiments, the vector comprises at least one T4-terminator (derived from bacteriophage T4). The T4-terminator can efficiently inhibit transcription and prevent readthrough from the integrase gene to the gene of interest. In some embodiments, the vector comprises a T4-terminator behind the expression cassette in the opposite direction to prevent further putative antisense readthrough. For example, the vector can comprise at least one T4-terminator before the expression cassette and / or at least one T4-terminator after the expression cassette. In some embodiments, the vector can comprise three terminators, one before and two after the expression cassette.

[0153] 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 further comprises an attachment site attP. The integrase of the φC31 integrase gene int mediates the integration of the vector into the host chromosome at different genomic positions by catalyzing the targeting and unidirectional recombination of two attachment sites: attP located on the vector and attB located on the host chromosome of the gene ACSP50_6589 (formerly 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) derived from attP-attB recombination (te Poele, Bolhuis and Dijkhuizen, 2008).

[0154] In some embodiments, the vector comprises an origin of transfer, such as the IncP origin of transfer, and / or a relaxosome gene, such as the relaxosome gene traJ. In some of these embodiments, the origin of transfer, such as the IncP origin of transfer, and / or the relaxosome gene, such as traJ, is derived from pSET152. The origin of transfer and the relaxosome gene enable transfer of the plasmid from the donor strain (e.g., Escherichia coli ET12567 / pUZ8002 (Kieser et al., 2000)).

[0155] In some embodiments, the vector according to the first aspect comprises an origin of replication, such as a high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication (ori). In some of these embodiments, the origin of replication, such as a high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication (ori), is derived from pSET152. The origin of replication, such as a high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication (ori), enables replication of the plasmid in the cloning strain (Escherichia coli DH5αMCR) and the donor strain (Escherichia coli ET12567 / pUZ8002).

[0156] In some embodiments, the vector according to the first aspect comprises at least one resistance marker, for example, a resistance marker (aac(3)IV, apmR) that mediates apramycin resistance. The resistance marker (aac(3)IV, apmR) that mediates apramycin resistance can be used for selection.

[0157] According to some embodiments of the fourth aspect, the expression vector comprises (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) at least one element of pSET152, such as a high-copy-number ColE1 / pMB1 / pBR322 / pUC according to SEQ ID NO: 89, and further does not comprise the putative antisense promoters according to SEQ ID NO: 108 and SEQ ID NO: 109.

[0158] 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 transfer origin (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 high-copy number ColE1 / pMB1 / pBR322 / pUC, the origin of replication (ori) according to SEQ ID NO: 89, and (e) at least one resistance marker arbitrarily, such as aac(3)IV, apmR, etc. that mediate apramycin resistance according to SEQ ID NO: 90, and (f) at least one T4-terminator arbitrarily, and (g) optionally, where the vector does not contain the putative antisense promoter according to SEQ ID NO: 108 and / or SEQ ID NO: 109.

[0159] According to some embodiments, the vector comprises the sequence described in SEQ ID NO: 110 or SEQ ID NO: 111. According to some embodiments, the vector comprises the sequence described in SEQ ID NO: 110 or SEQ ID NO: 111, or a fragment thereof.

[0160] In some embodiments, the vector is excellent by an easy cloning mechanism that allows the integration of different promoters. Thereby, the system can be rapidly adapted to further species, such as the acarbose-producing strain. In one aspect, the present invention provides the following. [Item 1] A method of engineering a Streptomyces strain, preferably an Actinoplanes strain, for improved production of acarbose, the method comprising engineering the Streptomyces strain (i) such that expression of the extracellular small carbohydrate-binding protein Cgt according to SEQ ID NO: 20 is lost or attenuated, and / or (ii) such that expression of at least one gene essential for carotenoid synthesis is lost or attenuated, and / or (iii) such that dTDP-D-glucose-4,6-dehydratase AcbB according to SEQ ID NO: 13 is overexpressed, and / or (iv) such that UDP-glucose-1P uridyltransferase GtaB according to SEQ ID NO: 19 is overexpressed The method as described above. [Item 2] The method according to item 1, wherein the method comprises (i) deletion or mutation of the gene encoding the extracellular small carbohydrate-binding protein Cgt according to SEQ ID NO: 20, and / or (ii) deletion or mutation of at least one gene essential for carotenoid synthesis, and / or (iii) introduction of a vector containing an expression cassette for AcbB into the Streptomyces strain, and / or (iv) introduction of a vector containing an expression cassette for GtaB into the Streptomyces strain The method as described above. [Item 3] The method according to item 2, wherein the expression cassette according to (iii) and / or (iv) is under the control of a moderately strong promoter characterized by a normalized glucuronidase activity of at least 1×10−4 [L·g−1·min−1] in a glucuronidase assay, or a strong promoter characterized by a normalized glucuronidase activity of at least 5×10−4 [L·g−1·min−1] in a glucuronidase assay. The method as described above. [Item 4] A Streptomyces strain, preferably an Actinoplanes strain, for producing acarbose, wherein said Streptomyces strain has been genetically engineered for the deletion or attenuated expression of the extracellular small carbohydrate-binding protein Cgt according to SEQ ID NO: 20. [Item 5] The Streptomyces strain according to item 4, wherein the Streptomyces strain is a cgt deletion mutant. [Item 6] A Streptomyces strain, preferably an Actinoplanes strain, for producing acarbose, wherein said Streptomyces strain has been genetically engineered for the deletion or attenuated expression of at least one gene essential for carotenoid synthesis. [Item 7] A Streptomyces strain, preferably an Actinoplanes strain, for producing acarbose, wherein said Streptomyces strain has been genetically engineered for the overexpression of dTDP-D-glucose-4,6-dehydratase AcbB according to SEQ ID NO: 13. [Item 8] A Streptomyces strain, preferably an Actinoplanes strain, for producing acarbose, wherein said Streptomyces strain has been genetically engineered for the overexpression of UDP-glucose-1P uridyltransferase GtaB according to SEQ ID NO: 19. [Item 9] At least one gene essential for carotenoid synthesis is a. Genes of the MEP / DOXP pathway i. 1-deoxy-D-xylulose-5-phosphate synthase gene dxs, ACSP50_7096 according to SEQ ID NO: 23, ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG, ACSP50_7248 according to SEQ ID NO: 24, iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr, ACSP50_7250 according to SEQ ID NO: 25, iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH, ACSP50_7707 according to SEQ ID NO: 26, v. 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE, ACSP50_7802 according to SEQ ID NO: 27, vi. 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase gene ispF, ACSP50_8046 according to SEQ ID NO: 28, and / or vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD, ACSP50_8047 according to SEQ ID NO: 29 b. Genes of terpene cluster 1 i. Isopentenyl-diphosphate delta-isomerase gene idi, ACSP50_0146 according to SEQ ID NO: 30, ii. Zeta-carotene desaturase gene crtI, ACSP50_0147 according to SEQ ID NO: 10, iii. Polyprenyl synthetase gene crtE / ldsA, ACSP50_0148 according to SEQ ID NO: 31, iv. Phytoene synthase gene crtB, ACSP50_0149 according to SEQ ID NO: 32, v. Deoxyribodipyrimidine photolyase gene, ACSP50_0150 according to SEQ ID NO: 33, or vi. Pyridine nucleotide-disulfide oxidoreductase gene, ACSP50_0151 according to SEQ ID NO: 34, c. Genes of terpene cluster 2a i. Transcription regulator gene ACSP50_1631 according to SEQ ID NO: 35, ii. Lycopene cyclase gene ACSP50_1632 according to SEQ ID NO: 36, iii. Lycopene cyclase gene ACSP50_1633 according to SEQ ID NO: 37, iv. Polyprenyl synthetase, farnesyl pyrophosphate synthetase 2 gene fps2 / crtE, ACSP50_1634 according to SEQ ID NO: 38, or v. Methylene tetrahydrofolate reductase (NADPH) gene, ACSP50_1635 according to SEQ ID NO: 39, d. Genes of terpene cluster 2b i. LysR-family transcription regulator gene, ACSP50_1650 according to SEQ ID NO: 40, ii. Methyltransferase type 11 gene, ACSP50_1651 according to SEQ ID NO: 41, iii. CDP-alcohol phosphatidyltransferase pgsA, ACSP50_1652 according to SEQ ID NO: 42, iv. Zeta-carotene desaturase (crtI-family) gene crtD, ACSP50_1653 according to SEQ ID NO: 43, v. Glycosyltransferase gene cruC, ACSP50_1654 according to SEQ ID NO: 44, vi. Hypothetical protein (put.membrane prot,) gene cruF, ACSP50_1655 according to SEQ ID NO: 45, vii. GCN5-family acetyltransferase gene, ACSP50_1656 according to SEQ ID NO: 46, viii. Monooxygenase gene, ACSP50_1657 according to SEQ ID NO: 47, or ix. Short-chain dehydrogenase gene, ACSP50_1658 according to SEQ ID NO: 48, e. Polyprenyl synthetase gene crtE, ACSP50_3873 according to SEQ ID NO: 49, or f. Genes of the camphor-like monoterpene biosynthesis terpene cluster 3 i. Transcription regulator (Crp / Fnr family) gene eshA, ACSP50_1949 according to SEQ ID NO: 104, ii. Camphene synthase gene, ACSP50_1950 according to SEQ ID NO: 50, iii. Methyltransferase (SAM-dependent) type 11 gene, ACSP50_1951 according to SEQ ID NO: 105, iv. Glycosyl-hydrolase gene, ACSP50_1952 according to SEQ ID NO: 106, or v. Oxidoreductase / ald / keto reductase, ACSP50_1953 according to SEQ ID NO: 107 An actinomycete strain for the production of acarbose according to item 6 or the method according to item 1, 2 or 3, comprising at least one gene selected from any of the above. [Item 10] An expression vector for Actinoplanes comprising an expression cassette for AcbB according to SEQ ID NO: 13 and / or an expression cassette for GtaB according to SEQ ID NO: 19, and / or an expression cassette for MerR according to SEQ ID NO: 22. [Item 11] Furthermore, a medium-strength promoter characterized by a normalized glucuronidase activity of at least 1×10-4 [L·g-1·min-1] in the glucuronidase assay, or a strong promoter characterized by a normalized glucuronidase activity of at least 5×10-4 [L·g-1·min-1] in the glucuronidase assay, the expression vector according to item 10. [Item 12] The expression vector according to item 10 or 11, further comprising a. φC31 integrase gene int according to SEQ ID NO: 85, and b. Transfer origin (incP) according to SEQ ID NO: 87, and c. Relaxosome gene traJ according to SEQ ID NO: 88, and d. Replication origin, Preferably, a high-copy number ColE1 / pMB1 / pBR322 / pUC, replication origin (ori) according to SEQ ID NO: 89 and e. Optionally, at least one resistance marker Preferably, a resistance marker mediating apramycin resistance, more preferably aac(3)IV, apmR according to SEQ ID NO: 90, and f. Optionally, at least one T4-terminator, comprising And optionally, wherein the vector does not contain the putative antisense promoter according to SEQ ID NO: 108 and / or SEQ ID NO: 109, said expression vector. [Item 13] The expression vector according to Item 11 or 12, wherein the strong promoter is a. apm according to SEQ ID NO: 96, b. ermE* according to SEQ ID NO: 98, c. katE according to SEQ ID NO: 94, d. moeE5 according to SEQ ID NO: 95, or e. gapDH according to SEQ ID NO: 82 selected from the group consisting of and / or the moderately strong promoter is f. efp according to SEQ ID NO: 92, g. cdaR according to SEQ ID NO: 97, h. rpsL according to SEQ ID NO: 99, i. rpsJ according to SEQ ID NO: 93, j. cgt according to SEQ ID NO: 91, or k. tipA according to SEQ ID NO: 81 selected from the group consisting of, said expression vector. [Item 14] An actinomycete strain for the production of acarbose according to any one of Items 4 to 9, wherein the strain contains the vector according to any one of Items 10 to 13, said actinomycete strain. [Item 15] Use of the actinomycete strain according to any one of the preceding claims in the production of acarbose.

Example

[0161] General tools and methods Strains and plasmids All strains used in this study are listed in Table E1. Recombinant strains used or created in this study are listed in Tables E2, E3, and E4 (Table E2 for plasmid-based expression systems, Table E3 for deletion and integration constructs cloned and stored in Escherichia coli DH5αMCR, and Table E4 for deletion and integration mutants of Actinoplanes species SE50 / 110).

[0162]

Table 3

[0163]

Table 4

[0164]

Table 5

[0165]

Table 6

[0166] Media and culture conditions Unless otherwise specified, all chemicals and culture 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).

[0167] Preparation of glycerol stock of Actinoplanes sp. SE50 / 110 For the preparation of glycerol stocks, Actinoplanes sp. SE50 / 110 (ATCC 31044) was grown in 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 with 86% (v / v) sterile glycerol in a 2:3 ratio. The glycerol stocks were stored at -80 °C.

[0168] Growth on solid medium and preparation of spore solution For sporulation, 200 - 300 μL of the glycerol stock was grown on a soybean flour medium (SFM - agar) (20 g·L -1 soybean flour (SOBO® Naturkost (Cologne, Germany)), 20 g·L -1 D - mannitol, 20 g·L -1 Bacto™ agar (Becton - Dickinson, Heidelberg, Germany), 167 μL of 10 N NaOH in tap water). Spores could be harvested by washing them off with 3 ml of ddH2O using a cotton swab after incubation at 28 °C for 5 - 7 days as described by Wolf et al. (2016).

[0169] 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 5 g·L of each K2HPO4 and KH2PO4 in ddH2O -1 to prepare a phosphate buffer consisting of) and filter sterilization was performed according to the protocol of Wendler et al. (2013).

[0170] Regarding the substitution of the carbon source maltose, instead of maltose monohydrate, 79.2 g·L -1 glucose·1H2O, 72.0 g·L -1 C-pur (Cerestar 01908, Cerestar GmbH, Krefeld, Germany), 71.9 g·L -1 galactose, 68.4 g·L -1 cellobiose, 71.9 g·L -1 D-arabinose, 72.0 g·L -1 D-lactose were each used. Mixtures of maltose and glucose were prepared at ratios of 90:10, 80:20 and 50:50 (v / v).

[0171] For the starch medium, a 4% (w / v) milky white solution of "starch soluble" from Acros Organics (part of Thermo Fisher Scientific, Geel, Belgium) was produced. For this, sterile water was preheated to 90 °C in a water bath and the weighed portion of starch was added with stirring. Subsequently, the remaining medium components were added. To enable comparison with the starch culture, an equivalent C molar concentration (here 44.4 g·L -1We prepared minimal media of maltose·1H2O (net weight of maltose). According to our research, by adding modifiers (HCl or NaOH), by adding inositol that is not metabolized, and by changing the concentration of the carbon source maltose respectively, we prepared media with various pH and osmotic pressures (data not shown).

[0172] Furthermore, minimal media with "starch soluble" from Acros Organics at 1 g·L -1 , 2 g·L -1 , 3 g·L -1 , 4 g·L -1 and 5 g·L -1 were prepared for culturing with limited carbon sources.

[0173] The pH and osmotic pressure of all media were determined according to the manufacturer's instructions by a pH meter Calimatic from Knick GmbH (Berlin, Germany) and an Osmomat 3000 from Gonotec GmbH (Berlin, Germany).

[0174] Shake flask culture Culturing was carried out in a 250 mL Corning® Erlenmeyer cell culture flask with baffles at 28 °C and 140 rpm for 7 days in a GFL shaking incubator 3032 or 3033 (Burgwedel, Germany). For inoculating 50 mL of the medium, 1 mL of a spore solution with OD = 3 - 5 was used. The cell dry weight was determined as described by Wolf et al. (2017a). The supernatant was stored at -20 °C for later analysis.

[0175] Microscale culture in the BioLector system of m2p-labs GmbH (Baesweiler, Germany) The comparative growth experiment was performed in a 48-well FlowerPlate covered with a gas-permeable sealing foil (m2p-labs GmbH, Baesweiler, Germany) with a volume of 1 ml and incubated at 28 °C and 800 rpm for one week with m2p-labs' RoboLector®. Growth was recorded by the backscatter signal. For the measurement of the final cell dry weight, 800 μL from each well was sampled into a weighed reaction tube (14,000 g, 2 min), washed with deionized water, and dried at 60 - 70 °C for one day. The supernatant was stored at -20 °C for later analysis.

[0176] Recombinant DNA research Unless otherwise specified, plasmid construction and assembly were performed by Gibson Assembly (Gibson et al., 2009). Fragments were amplified by PCR (Phusion® High-Fidelity PCR Master Mix with GC Buffer, NEB, Ipswich, MA, USA) in an Eppendorf thermocycler vapo.protect (Hamburg, Germany) and, if necessary, treated with DpnI (Thermo Fisher Scientific, Waltham, MA, USA). Purification of PCR products and gel extracts was performed by using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Dueren, Germany). Equimolar amounts of DNA fragments were added to the Gibson Assembly Master Mix at a ratio of 1:4. The master mix contained 0.64 μL of T5 exonuclease (10 U·μL -1 , NEB, Ipswich, MA, USA), 20 μL of Phusion High-Fidelity DNA polymerase (2 U·μL -1, Thermo Fisher Scientific, US), 160 μL of Taq DNA ligase (NEB, Ipswich, MA, USA), 699.36 μL of distilled water, and 320 μL of isothermal reaction buffer (25% PEG-8000, 1 mL of 1 M Tris-HCl, 100 μL of 1 M MgCl2, 100 μL of 1 M DTT, 20 μL of each 1 mM dNTP, 200 μL of NAD). The samples were incubated at 50 °C for at least 1 hour and subsequently transferred to Escherichia coli DH5αMCR by chemical transformation according to the protocol of Beyer et al. (2015). The selection of E. coli was carried out on 15 g·L -1 agar medium (Carl Roth, GmbH&Co.KG, Karlsruhe, Germany) and Luria / Miller broth medium containing 50 mg·L -1 of apramycin sulfate. Positive colonies were tested by PCR and gel electrophoresis, as well as Sanger sequencing by our in-house sequencing core facility.

[0177] Construction of plasmids for the gusA reporter system For the construction of plasmids for the gusA reporter system, see Schaffert et al. (2019).

[0178] Construction of a novel pSETT4 expression system For the cloning of the novel pSETT4 expression system, the pSET152 vector of Bierman et al. (1992) was used as a template. The vector backbone was linearized by PCR (Table E5).

[0179] A cloning cassette consisting of a gapDH-promoter under the control of a gapDH-promoter, a lacZ-gene under the control of a lac-promoter, and several restriction sites flanked by three T4 terminators was arranged as string DNA by Integrated DNA Technologies (Iowa, USA). Due to its complex structure, the cassette was ordered in three parts and assembled by GeneSOEing (Horton, 1995) using the primers in Table E5. Finally, the backbone and the insert were assembled by Gibson Assembly (Gibson et al., 2009). The new vector system was named pSETT4gap.

[0180] For the replacement of the gapDH promoter with the tipA promoter, pSETT4gap was digested with NdeI and KpnI and treated with shrimp alkaline phosphatase according to the supplier's instructions. All enzymes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The tipA promoter was amplified from pSETGUS (Myronovskyi et al., 2011) using 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 (see Figure 6).

[0181] [Table 7]

[0182] Overexpression of a single gene in the novel pSETT4 expression system For overexpression of a single gene, the insert was amplified by PCR (Table E6). The vector (pSETT4gap or pSETT4tip) was digested with BsaI (NEB, Ipswich, MA, USA) and assembled with the insert by Gibson Assembly (Gibson et al., 2009). For expression of the acbB gene under the control of the native promoter, the vector backbone pSET4gap was digested with BsaI and NdeI, leading to linearization of the vector under removal of the promoter. The gene of interest and the native promoter were amplified by using the primers in Table E6 and assembled with the vector backbone by Gibson Assembly (Gibson et al., 2009).

[0183]

Table 8

[0184] Construction of pCRISPomyces-2 deletion and integration vectors For construction of deletion and integration mutants by CRISPR / Cas9 technology, the plasmid pCRISPomyces-2 (Cobb et al., 2015) was used according to the protocol of Wolf et al. (2016). The spacer and its reverse complement were ordered as overlapping oligonucleotides from metabion GmbH (Steinkirchen, Germany) or Sigma-Aldrich (Taufkirchen, Germany) (Table E7).

[0185] The oligonucleotides were annealed into double strands and assembled with plasmids by Golden Gate Assembly (Engler et al., 2008) according to the protocol of Cobb et al. (2015). For the repair of Cas9-induced double-strand breaks, the DNA template was cloned into the vector backbone by Gibson Assembly (Gibson et al., 2009). As the DNA template, the flanking sequences upstream and downstream of the target gene (about 1 kB for each round) were amplified from genomic DNA by PCR (Table E8).

[0186]

Table 9

[0187]

Table 10

[0188] Deletion of the gene cgt by CRISPR / Cas9 technology For the construction of the Δcgt (ΔACSP50_5024) deletion mutant by CRISPR / Cas9 technology (clustered regular interspaced short palindromic repeats / CRISPR-associated endonuclease 9), plasmid pCRISPomyces-2 was used (Cobb et al., 2015). Spacer sequences were selected according to Wolf et al. (2016) and ordered as oligonucleotides with their reverse complements from Metabion GmbH (Steinkirchen, Germany) (spacer_1: 5’-acgcAGCGTCGCCCGCTGGGAGAA-3’, spacer_2: 5’-aaacTTCTCCCAGCGGGCGACGCT-3’). The oligonucleotides were annealed into double strands and assembled with the plasmid by Golden Gate Assembly (Engler et al., 2008) using BsaI (NEB, Ipswich, MA, USA) according to the protocol of Cobb et al. (2015). For the repair of Cas9-induced double-strand breaks, a deoxyribonucleic acid (DNA) template was cloned into the XbaI-linearized vector by Gibson Assembly (Gibson et al., 2009). As the DNA template, the flanking sequences upstream and downstream of the target gene (about 1 kB for each round) were amplified by polymerase chain reaction (PCR) using Phusion® High-Fidelity PCR Master Mix (NEB, Ipswich, MA, USA) with GC buffer (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, DNA fragments (flank_1: 1101 bp and flank_2: 982 bp) were added equimolarly at a ratio of 1:4 to a Gibson Assembly Master Mix consisting of 0.64 μL of T5 exonuclease (10 U / μL, NEB, Ipswich, MA, USA), 20 μL of Phusion High-Fidelity DNA polymerase (2 U / μL, Thermo Fisher Scientific, US) and 160 μL of Taq DNA Ligase (40 U / μL NEB, Ipswich, MA, USA), 699.36 μL of distilled water and 320 μL of isothermal reaction buffer (25% PEG-8000, 1 mL of 1 M Tris-HCl, 100 μL of 1 M MgCl2, 100 μL of 1 M DTT, 20 μL of each 1 mM dNTP, 200 μL NAD). After incubation at 50 °C for at least 1 hour, the reaction mixture was transferred to Escherichia coli DH5αMCR by chemical transformation according to the protocol of (Beyer et al., 2015). The growth and selection of Escherichia coli were carried out on Luria / Miller broth (LB medium) supplemented with 50 mg·L. -1 of apramycin sulfate in 15 g·L -1 They were plated on Luria / Miller broth (LB medium) containing agar medium KobeI (both: Carl Roth, GmbH&Co.KG, Karlsruhe, Germany). The plates were incubated at 37 °C for 10 - 14 hours. Apalomycin-resistant colonies were tested by PCR and gel electrophoresis for the first time and by Sanger sequencing by our in-house sequencing facility for the second time (PCR primer sequences: for: 5’-GGCGTTCCTGCAATTCTTAG-3’, rev: 5’-TCGCCACCTCTGACTTGAGC-3’, walking primers for sequencing: w1: 5’-CGCTGATCTTCAGCTTCC-3’, w2: 5’-GCCTTCACCTTCCATCTG-3’, w3: 5’-TCGGGAAAGCCGCCGGAG-3’)).

[0189] Conjugal transfer into Actinoplanes sp. SE50 / 110 Competent Actinoplanes sp. SE50 / 110 cells were prepared from freshly grown NBS cultures (see above). Cells were washed twice with 10% (w / v) ice-cold sucrose and twice with ice-cold 15% (v / v) glycerol. Finally, the cells were taken up in 15% (v / v) ice-cold glycerol (by adding approximately four volumes of cell pellet) and aliquoted into 100 μL in reaction tubes and snap-frozen in liquid nitrogen. Competent Actinoplanes cells are stored at -80 °C.

[0190] For conjugation, Escherichia coli ET12567 / pUZ8002 (Kieser et al., 2000) was used. The desired construct was transferred into Escherichia coli ET12567 / pUZ8002 according to Beyer et al. (2015), and then -1 on LB agar plates supplemented with 50 mg·L -1 of apramycin-sulfate, 50 mg·L -1 of kanamycin-sulfate and 15 mgL -1 of chloramphenicol, after selection, the cells were grown in liquid culture (LB-medium containing the same supplements) and harvested at an optical density of 0.4 - 0.6. The cells were washed twice with ice-cold LB medium and mixed with competent cells of Actinoplanes sp. SE50 / 110. The cell suspension was plated on SFM agar plates. After incubation at 28 °C for 20 - 24 h, 1 mL of 500 mg·L -1 of apramycin sulfate dissolved in ddH2O was dispensed onto the plate with a sterile cotton swab. The first exconjugants of Actinoplanes sp. SE50 / 110 can be observed after one week. The exconjugants were transferred to SFM agar plates supplemented with 50 mg·L -1 of apramycin-sulfate. To purify Actinoplanes exconjugants of E. coli origin, repeated streaking was performed several times. To facilitate this process, 50 mg·L -1 of phosphomycin or trimethoprim can be added to the medium to remove the donor strain.

[0191] Plasmid curing to obtain marker-free CRISPR / Cas9 deletion / insertion mutants of Actinoplanes sp. SE50 / 110 Plasmid curing was performed by culturing in complex medium NBS at high temperature according to the protocol of Wolf et al. (2016). 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 deletions by PCR (primer sequence data not shown). PCR fragments were excised from the gel and sequenced by our in-house Sanger sequencing core facility.

[0192] Furthermore, to rule out off-target effects, genomic DNA of deletion or integration mutants was also sequenced by Oxford Nanopore Technologies (Oxford, UK). For this, genomic DNA of NBS growth cultures was isolated using the NucleoSpin® Microbial DNA kit (Macherey-Nagel, Dueren, Germany). Libraries were prepared with the help of 1D genomic DNA by the ligation kit (Oxford Nanopore, Oxford, UK).

[0193] Deletion system based on homologous recombination with cytosine deaminase CodA and counterselection Vector integration into the genes of the acb gene cluster is occurring by the use of the replicative vector pKC1139. Based on this observation, a novel deletion system using homologous recombination was developed and tested by way of the example of the gene cgt (ACSP50_5024).

[0194] Conjugation into Actinoplanes sp. SE50 / 110 was enabled using a vector backbone with the transfer origin (ncP) and the relaxosome gene traJ. In this study, two different antimicrobial resistance markers mediating apramycin and kanamycin resistance: namely aph(3’)II (kan R , kanamycin) and aac(3)IV (apm R, apramycin) were tested for selection. Furthermore, high-copy number ColE1 / pMB1 / pBR322 / pUC origins of replication were incorporated to enable replication in the donor strain E. coli. ori, oriT ncP , the tra gene and the resistance cassette were taken from pRT802 and pRT801 (Gregory et al., 2003), respectively. In Actinoplanes sp. SE50 / 110, neither the replicon for replication nor the integrase gene with an attachment site is included in the new deletion system, so the vector can only be maintained in Actinoplanes sp. SE50 / 110 when integrated into the genome by homologous recombination (Figure 7). For this purpose, a 2-kB homologous sequence adjacent to the gene cgt was incorporated. After conjugative transfer in Actinoplanes sp. SE50 / 110, mutants in which the first crossover occurred can be selected by apramycin or kanamycin resistance. To force the excision of the vector backbone (the second crossover), 5-fluorocytosine (5-FC) was added, which is converted by the cytosine deaminase CodA to the toxic product 5-fluorouracil (5-FU). In this study, codA codon-optimized for Streptomyces ssp was used (Dubeau et al., 2009). After the second crossover, either the wild-type genotype or the genotype of the deletion mutant is present.

[0195] The new deletion system was successfully tested for the gene cgt as shown by colony PCR and ONT-sequencing. The proportion of deletion mutants after the second crossover was successful was 25 - 32%. The workflow is shown in Figure 8.

[0196] Analytical methods Quantification of acarbose from the supernatant by high performance liquid chromatography (HPLC) The supernatant of a maltose-grown culture of Actinoplanes sp. was 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 on an Agilent HPLC system 1100 series (G1312A Binary Pump Serial#DE43616357, G1329A ALS Autosampler Serial#DE43613 / 10, G1315A Diode Array Detector (DAD) Serial#DE72002469). As stationary phase, a Hypersil APS-2 column (125 × 4 mm, 3 μm particle size) from Thermo Fisher Scientific Inc. (Waltham, MA, USA) was used, heated to 40 °C. As mobile phase, 1 mL min -1 of 68% acetonitrile (solvent B) and 32% phosphate buffer (0.62 g L -1 KH2PO4 and 0.38 g L -1 An isocratic flow of Na2HPO4·2H2O (solvent A) was applied. 40 μL of each sample was injected and separated over a 10 min run. Acarbose was detected using a DAD detector at 210 nm (reference 360 nm) and quantified from the peak area of the calibration curve.

[0197] Liquid chromatography-mass spectrometry (LC-MS) Preparation of samples for analysis of intracellular metabolites Triplicates of Actinoplanes sp. strain SE50 / 110 were grown in maltose minimal medium for at least 4 days. 10 mL of culture was rapidly filtered through filter paper in a Büchner funnel to obtain 2.63 g L -1It was washed with an aqueous NaCl solution. The cells were transferred to a pre-weighed round-bottom screw-cap tube, rapidly frozen in liquid nitrogen, and stored at -80 °C. The cells were dried overnight using a centrifugal evaporator (SpeedVac) from Thermo Fisher Scientific (Waltham, MA, USA). 4 mg of the dried cells were transferred to a round 2 mL screw-cap tube (Bio Spec Products Inc., Bartlesville, USA) containing a mixture of zirconia / silica microbeads (about 500 μL) sized 0.1 mm, 0.05 mm, and 0.01 mm, and 700 μL of 80% MeOH was added to the cells and beads. Cell disruption was performed 3 times for 30 seconds at a speed setting of 6.5 in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA). The sample was cooled on ice for 5 minutes during that time. The cell suspension was centrifuged at 13,000 g and 4 °C for 5 minutes. 500 μL of the supernatant was transferred to an HPLC vial, dried under a nitrogen stream, and taken up in 50 μL of distilled water.

[0198] Sample preparation for the analysis of extracellular acarbiosyl metabolites Sample preparation was carried out according to the protocol described by Ortseifen (2016). Sugars and pseudosugars were concentrated from 10 mL of the supernatant by solid-phase extraction using a Chromabond® Easy column (Macherey-Nagel, Dueren, Germany, REF 730753). The column was equilibrated with 3 mL of methanol and then washed with 3 mL of distilled water before loading the sample. Nonspecifically bound metabolites were washed with 3 mL of 95% (v / v) methanol. Elution was performed with 3 mL of methanol.

[0199] LC-ESI-MS of intracellular and extracellular metabolites For LC-MS, a LaChromUltra (Hitachi Europe Ltd., UK) HPLC system connected to a microTOF-Q hybrid quadrupole / time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) source was used.

[0200] For the analysis of intracellular metabolites, 2 μL of the sample was separated on a 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) and eluent B (acetonitrile) were applied at a flow rate of 0.2 mL·min -1 using the following gradient: 0 min B: 90%, 30 min B: 25%, 37.5 min B: 25%, 40.0 min B: 80%.

[0201] As standards for peak identification, 2 μL of 10 μM UDP-glucose, glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, and dTDP-glucose were injected.

[0202] For the analysis of extracellular acarbose metabolites, 10 μL of the sample was separated on a 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, and 0.1% (v / v) formic acid) and eluent B (90% (v / v) acetonitrile, 10% (v / v) H2O, and 0.1% (v / v) formic acid) were applied at a flow rate of 0.4 mL·min -1 using the following gradient: 0 min B: 100%, 8 min B: 0%, 13 min B: 0%, 15.5 min B: 100%, 18 min B: 100%.

[0203] The ESI source was operated in negative ionization mode for the analysis of intracellular metabolites and in positive ionization mode for the analysis of extracellular acarbose metabolites. The temperatures of the drying gas and the capillary were set at 180 °C. The MS scan range was set at 200 - 1,000 m / z (intracellular metabolites) and 50 - 3,000 m / z (extracellular acarbose metabolites), respectively.

[0204] The peak areas of specific masses were integrated using the software Compass™ (Bruker Daltonics, Bremen, Germany). The peaks were normalized by the weighed amount of dry cells (intracellular metabolites) and the dry cell weight at sampling (extracellular acarbose metabolites), respectively.

[0205] Extraction and analysis of carotenoids Extraction Cell pellets from Actinoplanes sp. SE50 / 110 were transferred to a 2 mL screw-cap tube together with approximately 500 μL of a mixture of zirconia / silica microbeads (Bio Spec Products Inc, Bartlesville, USA) of sizes 0.1 mm, 0.05 mm and 0.01 mm. 1 mL of acetone or methanol was added as an extraction solvent. Cell disruption was carried out three times for 45 seconds at a speed setting of 6.5 in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA). The samples were cooled on ice for 5 minutes during that time. The homogenized cell suspension was centrifuged at 13,000 g and 4 °C for 20 minutes. The supernatant was transferred to a glass vial. For HPLC analysis, a mixture of the acetone extract and the methanol extract was produced at a ratio of 7:3 and transferred to a new glass vial.

[0206] 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 of petroleum, 11 mL of isopropanol and 50 μL of water. The operation was carried out in the dark. After drying the TLC plate, the bands were scraped off with a scalpel and transferred to a new tube. After adding 1 mL of ethanol, the absorption spectrum was analyzed using a Genesys 10S UV-Vis spectrophotometer from Thermo Fisher Scientific (Waltham, MA, USA).

[0207] HPLC Analysis of Carotenoids by Absorbance Scanning The carotenoids were separated by reversed-phase HPLC using an Agilent 1200 series HPLC system (Agilent Technologies GmbH & Co. KG, Boeblingen, Germany) equipped with a diode array detector (DAD) for UV-Vis spectra, following Henke et al. (2017) and Heider et al. (2014). A 20 μL sample volume was applied at a flow rate of 0.5 mL·min -1 as described previously (Heider et al., 2014; Henke et al., 2017), 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 as the stationary phase.

[0208] The following gradient was applied: 0 min A: 100%, 32 min A: 75%, 47 min A: 0%, 70 min A: 0%, 75 min A: 100%. Eluent A consisted of 0.1 M ammonium acetate in deionized water and methanol in a ratio of 15:85 (v / v). Eluent B consisted of a mixture of methanol, acetonitrile and acetone in a ratio of 44:43:13 (v / v). Detection of carotenoids was performed at 470 nm. In addition, a wavelength scan between 360 nm and 700 nm was performed every second during operation.

[0209] Assay Promoter Screening Experiment by Spectrophotometric Measurement of Glucuronidase Activity Two different types of glucuronidase assays were performed: one using crude protein extracts and one using whole cells. The protocols described by Horbal et al. (2013) and Siegl et al. (2013) were adapted for Actinoplanes sp. SE50 / 110. Since the substrate p-nitrophenyl-D-glucuronide was found to dissociate under our assay conditions, the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc, AppliChem GmbH, Darmstadt, Germany) was selected.

[0210] Growth Conditions and Sample Preparation Actinoplanes mutants with promoter constructs carrying the gusA gene were cultured in maltose minimal medium for 1 week as described above. The assays were performed during the growth phase. 500 μL of each culture was sampled for the assay using whole cells. 1 mL was sampled for the assay using crude protein extracts and transferred to screw-cap tubes containing zirconia / silica microbeads (Bio Spec Products Inc., Bartlesville, USA) of 0.1 mm and 0.05 mm sizes. Cells were disrupted in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) at speed setting 6.5 for 2 times for 30 seconds each (with 5 minutes on ice in between). After centrifugation, the lysate was transferred to a new reaction tube and centrifuged again. The supernatant was used for the cell-free assay. Total protein quantification was performed by Bradford assay (see above).

[0211] Glucuronidase (gus) Assay The GUS assay was performed in black microtiter plates (96-well PS F-bottom μCLEAR, black, med.binding, Greiner Bio-One, Kremsmuenster, Oesterreich, REF 655096). 100 μL of each sample (either cell suspension or lysate) was pipetted into three wells, one of which served as a 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), 5 mM DTT and 0.1% Triton-X-100) was supplemented with 2 mM substrate X-Gluc (stock solution: 0.2 M in DMF). 100 μL was added to the 100 μL of the sample. For the negative control, 100 μL of GUS buffer without substrate was added. In addition to the individual negative controls for each sample, medium and substrate controls were also prepared.

[0212] The microtiter plates were measured in a pre-warmed Tecan reader Infinite M200 (Ref 30016056, Tecan Group AG, Maennedorf, Switzerland) (37 °C) for 3 hours (assay using whole cells) and 2 hours (assay using lysates), respectively. The absorption maxima of indigo were measured at 610 and 660 nm. After subtracting the absorption values of all controls, the slope of each absorption curve was calculated by linear regression and normalized by either cell dry weight (assay with whole cells) or total protein amount (assay with lysates). The β-glucuronidase activities in different mutants were compared using the normalized slope. Screening Experiment in the Biolog® OmniLog Phenotype Microarray System To evaluate respiration on different carbon sources (panels PM1 and PM2), a prescreening experiment was conducted in a Biolog® OmniLog Identification System (Hayward, CA, USA). The actinomycetes species SE50 / 110 wild type and deletion mutant Δcgt were grown on SFM agar plates as described elsewhere in this specification. Cells were collected by using a sterile cotton swab and diluted with the inoculation fluid IF-0a for PM1 and PM2. The turbidity of the cell suspension was checked in a Biolog® turbidimeter to achieve 80% transmittance according to the manufacturer's protocol. 2.32 mL of the cell suspension was added to 20 mL of IF-0a, 0.24 mL of 0.5 M MgCl2, 0.24 mL of 0.5 M Na2SO4, 0.24 mL of 0.5 M NH4Cl, 0.24 mL of 1.0 M Na3PO4, 0.24 mL of distilled water, 0.24 mL of Biolog redox dye mix G, and 0.24 mL of a metal ion cocktail (each 5.0 mM: ZnCl2·7H2O, FeCl2·6H2O, MnCl2·4H2O, CaCl2·2H2O) according to the manufacturer's protocol. The PM panel was inoculated with 100 μL / well of the prepared solution and incubated at 28 - 30 °C for 1 week in an OmniLog system (Mode 71000 Serial#406). Data evaluation was performed using the manufacturer's software (Kinetic Analysis, Biolog and Omnilog 2.3, Biolog).

[0213] RNA Research Sampling and RNA Isolation For transcriptome analysis, 2 × 1 mL cultures were taken during the growth phase, separated from the supernatant by centrifugation (10 s), and snap-frozen in liquid nitrogen. The pellet was stored at -80 °C until further processing.

[0214] For the isolation of ribonucleic acid (RNA), the frozen cell pellet was resuspended in 500 μL of LB buffer (NucleoSpin® RNA Plus, Macherey-Nagel, Dueren, Germany) and transferred to a 2 mL lysis matrix tube (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA). Cell disruption was performed three times for 20 seconds at a speed setting of 6.5 in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) (with 5 minutes on ice in between). Subsequently, the cell suspension was centrifuged at 13,000 g and 4 °C for 5 minutes. The supernatant was used for RNA extraction using the NucleoSpin® RNA Plus kit combined with rDNase Set (Macherey-Nagel, Dueren, Germany) for on-column DNA digestion. After washing and elution according to the manufacturer's protocol, DNA digestion was repeated (in solution) and the samples were washed again using the same kit. The samples were tested for residual DNA using two primer pairs that bind to the genomic DNA of Actinoplanes sp. SE50 / 110 and amplify small fragments of about 200 - 300 nt. If necessary, DNA digestion and RNA clean-up were repeated. The amount of RNA was analyzed using a NanoDrop 1000 spectrophotometer (Peqlab, Erlangen, Germany).

[0215] Reverse Transcription Quantitative PCR Reverse transcription quantitative PCR was performed in a LightCycler 96 System of Roche (Mannheim, Germany) using the SensiFast SYBR No-Rox One-Step kit (Bioline, London, UK) and a 96-well light cycler plate (Sarstedt, Nuembrecht, Germany) according to the protocol of Wolf et al. (2017a). The relative RNA amount was normalized against total RNA (100 ng), and 2 -ΔCqIt was calculated as follows. ΔCq is the difference in the average Cq in the mutant strain compared to the control strain. The primers in Table E9 were used for the determination of relative gene transcription.

[0216]

Table 11

[0217] Whole Genome Oligonucleotide Microarray The whole-genome oligonucleotide microarray was performed according to the protocol of Wolf et al. (2017a), which was adapted to the high G+C content of the Actinoplanes species SE50 / 110 for the hybridization procedure.

[0218] Triplicate RNAs were isolated and pooled equimolarly (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, CA, USA) was used according to the manufacturer's instructions with practical adjustments described by Wolf et al. (2017a). The Amersham CyDye monoreactive dye pack (GE Healthcare, Little Chalfont, UK) was used for labeling. A custom whole-genome oligonucleotide microarray representing the coding sequence of the actinomycete species SE50 / 110, which was designed by Wolf et al. (2017a) (4x44K format, representing 43,803 features of 8,238 genes and 1,417 control spots, supplier: Agilent Technologies, Santa Clara, CA, USA) was used. All microarray-specific reagents and devices, including hybridization oven and scanner, were used from Agilent Technologies (Santa Clara, CA, USA). Agilent Feature Extraction Software Version 10.7.3.1 (Agilent Technologies, Santa Clara, CA, USA) was used for feature extraction (protocol GE2_107_Sep09). Subsequent data analysis, including LOWESS normalization and statistical analysis, was performed using the Microarray and Gene Expression (MAGE)-compliant system EMMA 2 (Dondrup et al. 2009). A p-value of 0.05 was used as the significance cut-off. The M-value cut-off for a false discovery rate of 0.01 was determined to be 1.1 and -1.1 according to previous "yellow experiments" performed by Wolf et al. (2017a).

[0219] Analysis of the functional relevance of Cgt Distribution of Single Domain CBM-20 Proteins in the Eubacterial World The inventors analyzed the distribution of CBM-20 single-domain proteins in the prokaryotic world by BlastP analysis.

[0220] Briefly, the distribution of specific CBM-20 domain proteins was analyzed by BlastP analysis using the NCBI non-redundant protein database (Altschul et al., 2005; Altschul et al., 1990). Since the CBM-20 domain is present in various different proteins and enzymes, data filtering had to be carried out: among the first 3,316 BlastP hits, all of eukaryotic origin and those with a function-specific annotation or all enzymes with a size exceeding 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, a total of 53 proteins, contain two CBM-20 domains traversed by higher domains described as glycosyl-hydrolase-77-superfamily 4-alpha-glucanotransferase. Ten contain different additional domains: five of them are alpha-amylase inhibitor domains, two are CBM-25 and N-terminal CBM-26 binding domains respectively, two N-terminal domains of the IPT superfamily which probably have a regulatory function, and one is the DUF1393 domain which has been described as occurring in some alpha-amylases (information obtained from the NCBI database). These candidates were also excluded. Only 18 candidates (including Cgt from Actinoplanes sp. SE50 / 110) showed a specific CBM-20 domain. Based on the multiple sequence alignment performed by BlastP (Altschul et al., 1990; Altschul et al., 2005), a protein tree was created by the Blast tree view 1.17.5 of the NCBI database (NCBI database).

[0221] Interestingly, the specific CBM-20 domain protein was found only in 17 other species (Figure 9). Most of these are found in all strains of species in the order Actinomycetales, such as the genus Actinoplanes. Most of the 17 species were originally isolated from soil and environmental samples, namely, A. missouriensis (described by Parenti and Coronelli, 1979), A. utahensis (described by Parenti and Coronelli (1979) and first isolated by Couch (1963)), A. teichomyceticus (Wink et al., 2006), Streptomyces sp. 94 (Chu et al., 1996), Streptomyces sp. OK885 (isolated from roots, Tennessee, USA, information taken from GenBank (Benson et al., 2013) of the NCBI (NCBI database)), Streptosporangium roseum (Nolan et al., 2010), Streptosporangium sclerotialus (syn. Chainia antibiotica) (Thirumalachar, 1955), Cellulomonas sp B6 (Piccinni et al., 2016), Paenibacillus sp P22 (Hanak et al., 2014), Clostridium sp DMHC 10 isolated from sludge in a distillery waste treatment plant (Kamalaskar et al., 2010). The CBM-20 protein is also present in Streptomyces sp. DI166, where the sampling location is not reported, and in multiple species of the family Pseudomonadaceae. They belong to genera known to include members that inhabit soil.

[0222] Strains carrying the specific CBM-20 protein without a direct association with soil or the environment occur only rarely, similar to specific isolates of the human pathogens Chlamydia trachomatis (Thomson et al., 2008) and Mycobacterium abscessus (Ryan and Byrd, 2018; Moore and Frerichs, 1953).

[0223] Confirmation of Starch Binding Function by In Vitro Assay The CBM-20 domain has been described as having starch-binding function, and the inventors wished to test this by in vitro assay. The small carbohydrate-binding protein Cgt is highly expressed and concentrated in the extracellular space due to its N-terminal signal peptide (Wendler et al., 2015a), so the protein can be concentrated directly from the supernatant by filtration. Starch-binding assays were performed using different concentrations of potato-derived starch. Both the starch fraction and the supernatant were analyzed by SDS-PAGE. In all starch fractions (in the range of 1 - 10% (w / v) of starch), a protein band of approximately 15 kDa was detected, which was clearly identified as Cgt by MALDI-TOF-MS. In contrast, the supernatant fraction was almost completely depleted of 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 the Cgt remained in the supernatant fraction. In addition to Cgt, another small extracellular protein of unknown function, ACSP50_6253, was identified by the starch-binding assay (data not shown).

[0224] Analysis of cgt Expression during Growth on Different Carbon Sources The gene cgt has been reported to be differentially expressed in the presence of different carbon sources as determined by transcriptome and proteome analysis for glucose and maltose (Schwientek et al., 2013; Wendler et al., 2015a; Ortseifen, 2016). The inventors tested the effect of several carbon sources on the expression of the cgt gene by measuring the transcript levels by reverse transcription quantitative PCR (RT-qPCR). For this purpose, a 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) (Figure 10). The latter is a sugar-containing product from the degradation of starch consisting mainly of maltose and maltotriose. All carbon sources were supplemented at an equivalent C molar amount. The only exception was starch: due to its low solubility, here a 4% (w / v) milky white solution of "starch soluble" from Acros Organics was generated. In contrast, a maltose minimal medium with reduced maltose amount (here 44.40 g·L -1 maltose monohydrate) was prepared, where the C molar concentration should approximate that in the starch medium.

[0225] For most of the carbon sources tested, the transcription of the cgt gene was similar or only slightly different compared to the maltose-grown cultures and was not significantly decreased (Figure 11A). Differential transcription was observed to a small extent for galactose (3.4 less transcription, log2(fold change) = 0.291). A significant decrease in cgt transcripts was measured for the carbon sources glucose (142-fold less transcription, log2(fold change) = 0.007) and lactose (62-fold less transcription, log2(fold change) = 0.016). When the cells were grown on a maltose minimal medium with reduced maltose (here 72.06 g·L -1 instead of 44.4 g·L -1 ), a 2.9-fold decreased transcription of the cgt gene was observed (log2(fold change) = 0.345) (Figure 11B).

[0226] Analysis of Gene Deletion Mutation Δcgt Δcgt under Various Carbon Sources and Carbon Limitation Conditions The differential transcriptional profile of cgt dependent on the carbon source indicated functions within sugar metabolism as previously estimated (Ortseifen, 2016). Ortseifen (2016) suggested that Cgt is involved in the retention of carbon as an energy source in the context of the carbon for energy model. The growth of wild type and the CRISPR / Cas9 deletion mutant Δcgt was tested with different carbon sources in liquid culture.

[0227] Previously, prescreening experiments were carried out in the OmniLog Phenotypic Microarray System (Biolog Inc, Hayward, United States of America), which enabled rapid phenotypic screening by measuring cell respiration activity on a total of 190 different carbon sources in multiwell plates. Among these, Actinoplanes showed respiration on 103 carbon sources. Except for arabinose and lactose, no differential respiration profile of Δcgt was observed on the remaining 101 carbon sources. To verify these results regarding the level of growth, the carbon sources arabinose and lactose were further tested in shake flask cultures. Also, to mimic the natural carbon sources of the habitat soil, the standard laboratory sugars maltose and glucose, as well as the complex carbon sources starch and the disaccharide cellobiose were tested. No growth constraint was observed for Δcgt (Figures 12 and 13).

[0228] Furthermore, growth under carbon limitation conditions (here: 1 g·L -1 、2 g·L -1 、3 g·L -1 、4 g·L -1 、5 g·L -1 starch) was tested with the m2p-labs RoboLector®-system. No growth disadvantage of the cgt mutant was observed in case of carbon source limitation compared to the wild type (Figure 14). Δcgt Does Not Affect Osmotic or pH Tolerance It has been proposed that Cgt multimers form a surface layer by multimerization (Wendler et al., 2015a). This may suggest a potential role in defense against environmental changes such as drought, pH, and osmotic pressure. pH screening was performed on solid medium as in the culture medium of the RoboLector® system. For screening on solid medium, SFM-agar plates with pH in the range of pH 4 to 11 (in 1-step) were prepared, and droplets of a dilution series of spores of wild type and deletion mutant Δcgt were applied. Both the mutant and wild type were able to grow at pH 5 to 11. No differences in growth or sporulation on the agar plates were observed.

[0229] Since it is difficult to evaluate the effect of drought tolerance, the inventors analyzed colony and sporulation on the surface of the bacterial flora, and no differences were found between the wild type and Δcgt.

[0230] For pH screening in liquid culture, maltose minimal medium with pH in the range of 4 to 7 was prepared. Since the medium components tend to precipitate, higher pH values could not be tested in liquid culture. Both strains grew from pH 4.5 to 7 (Figure 15). No differences were observed regarding the final cell dry weight.

[0231] For osmotic pressure screening, maltose minimal media with various concentrations of maltose in the range of 3.6 to 108.1 g·L -1 of maltose monohydrate and osmotic pressure in the range of 323.5 to 681.0 mOsmol·kg -1 were prepared (Table E11). No significant growth differences were observed between the wild type and deletion mutant Δcgt (Figure 16).

[0232] In addition, inositol was tested as an osmotic pressure regulator since it is not consumed by Actinoplanes. Here, the osmotic pressure was 388.5 to 695.0 mOsmol·kg -1It was within the range of, but no growth difference was observed (Figure 17). 159 - 190 mOsmol·kg -1 Lower osmotic pressures between were tested by using the complex medium NBS (Figure 19, Table E10). Again, no significant difference in growth was observed between the wild - type strain and the deletion mutant Δcgt.

[0233]

Table 12

[0234] Δcgt Shows Improved Acarbose Production on Maltose Minimal Medium No clear growth phenotype could be observed under the test conditions, but the lack of highly expressed Cgt protein seems to conserve cellular metabolic resources such as ATP and amino acids. These could be used for cell growth or other assimilation processes. In the experiment, Δcgt did not show a significant growth advantage. However, a significantly higher final acarbose concentration was detected for the deletion mutant Δcgt compared to the wild - type (Table E10). In culture in complex medium, this was most prominent during the growth phase (Figure 18).

[0235] The improved acarbose - producing phenotype was confirmed by three independent shake - flask cultures in maltose minimal medium (Figure 19 and Table E11). Quantification of acarbose from the supernatant showed an improved acarbose yield coefficient for the deletion mutant compared to the wild - type. The difference in the final acarbose yield was significant (tested by two - sided t - test, p - value = 0.04608). As a result, an 8.3 - 16.6% increase in the final acarbose concentration was achieved in Δcgt (see Table E11).

[0236]

Table 13

[0237] Δcgt Does Not Affect the Expression of Acarbose Biosynthesis Genes Deletion of the highly expressed gene cgt did not negatively affect the growth or viability of the organism under various conditions, but the finding that an enhanced yield to the acarbose production phenotype was obtained was surprising. Thus, RT-qPCR of representative acb genes was also performed to exclude a direct effect on the regulation of the acarbose biosynthesis (acb) genes. For this purpose, wild type and Δcgt were grown on minimal maltose medium and RNA was isolated from samples of the early growth phase. The relative transcript levels of the acarbose biosynthesis cluster genes acbZ, acbW, acbV, acbA, acbB, acbD and acbE were calculated for Δcgt compared to the wild type (Figure 20). The acbV gene is the first of several polycistronic transcribed genes within the major operon of the acarbose biosynthesis gene cluster (Wolf et al. 2017b). The monocistronic transcribed genes acbD and acbE, which encode proteins for extracellular acarbose metabolism, have been shown to be strongly regulated by the acarbose regulator AcrC (Wolf et al., 2017a). The genes acbA, acbB and acbZ are also monocistronically transcribed and are annotated as enzymes for acarbose biosynthesis (acbAB) and its extracellular metabolism (acbZ), respectively. AcbW is the first gene of the acbWXY-operon and is presumed to encode an ABC transporter. For all selected transcripts, no significant change in relative transcript concentration was measured in the deletion mutant Δcgt compared to the wild type (Figure 20).

[0238] Discussion The relationship between carbohydrate metabolism and acarbose biosynthesis is very interesting. Recent studies have pointed out the importance of carbon utilization in the context of acarbose and further acarbiosyl metabolite biosynthesis in the wild type (Wendler et al., 2014).

[0239] In this context, the starch-binding protein Cgt is prominent. It is one of the most strongly expressed genes in Actinoplanes sp. SE50 / 110 (Schwientek et al., 2013) that constitutes approximately 8% of the entire secretome (unpublished data of the present inventors). Its gene product is transported extracellularly (Wendler et al., 2013). Overproduction and transport mean high costs for the cell: only for the translation process, 4 ATPs are required per peptide bond (Campbell and Reece, 2011; Purves, 2006), i.e., without including additional costs for RNA synthesis, amino acid production, protein folding and transport. Therefore, the present inventors concluded that Cgt has a significant role in Actinoplanes sp. SE50 / 110 physiology. Two different functions of Cgt: its role in carbohydrate metabolism and its role as a surface protein are proposed and analyzed herein.

[0240] For the starch-binding domain, Ortseifen (2016) suggested that Cgt may be involved in the binding and retention of energy sources in the context of the carbophore model (Wehmeier, 2003). Evidence was also provided by RT-qPCR showing differential expression of the gene cgt in cultures grown with glucose, galactose and lactose compared to cultures grown with maltose, higher maltodextrins and cellobiose. This follows differential proteome analysis for the carbon sources maltose and glucose (Wendler et al., 2015a; Wendler et al., 2015b). These results indicate carbon-dependent expression of cgt. It would be interesting to elucidate the regulatory mechanism. However, in Actinoplanes sp. SE50 / 110, more than 900 genes are estimated to be involved in transcriptional regulation, of which 697 are annotated as transcriptional regulators according to the annotation of Wolf et al. (2017b) (GenBank: LT827010.1).

[0241] The sugar-dependent expression of cgt may exhibit functions within the utilization of maltose, higher maltodextrins, and potentially cellobiose. However, our study of the deletion mutant Δcgt has not revealed phenotypic differences regarding carbon utilization. This was tested for a total of 105 different carbon sources, of which 103 were analyzed by the OmniLog screening system and 6 were analyzed in liquid culture.

[0242] The function of Cgt can be ignored under excess carbon sources, but is essential when growing under limited carbon source conditions. Therefore, the inventors tested the growth of the deletion mutant Δcgt and the wild type on minimal media containing low concentrations of starch. Starch was selected as the carbon source, and due to the starch-binding activity of Cgt, this was confirmed in the starch-binding assay here. Nevertheless, the growth phenotype of the mutant could not be observed under limited carbon source conditions.

[0243] Another function within sugar metabolism could lie in the binding of insoluble crystalline substrates, which can cause structural changes, increase substrate accessibility, and enhance the activity of other hydrolases such as amylases. Such mechanisms have already been described for the soil bacterium Serratia marcescens for chitin degradation (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-glycoside functions, and three of them, the α-amylase / pululanase AcbE, AcbZ, and PulA, have been shown to accumulate in the extracellular space (Wendler et al. 2015a). Furthermore, another small extracellular protein (ACSP50_6253) with unknown function and starch-binding ability was identified in the starch-binding assay. Through the heterologous expression of extracellular amylases and enzyme assays in the presence and absence of both -Cgt and ACSP50_6253-, a supporting function during starch degradation could be detected in future experiments.

[0244] Apart from its role in sugar metabolism, a function as a surface layer protein is also 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, one of which is involved in translocation via the Sec pathway as part of the leader peptide, and the second of which is hypothesized to be required for multimerization. Although Cgt is unlikely to be physically immobilized in the membrane (Wendler et al., 2015a), the Cgt protein may remain as a multimer in the hyphal mesh due to reduced fluid flow. In this context, the starch-binding domain may also serve as an anchor.

[0245] In its role as a putative surface protein, the inventors first hypothesized a protective function in the context of pH and osmolyte stress or drought situations. However, screening experiments showed that deletion of the cgt gene did not result in significant growth inhibition at different pH values in liquid cultures. Screening experiments on solid media gave no indication that Cgt may have a protective function in the case of pH or drought.

[0246] A hint of a function presumed to be related to osmoregulation was provided by reverse transcription quantitative PCR of wild type grown with different amounts of maltose. Here, the inventors observed a 2.9-fold decrease in the transcription of the gene cgt when growing on maltose compared to 72 g·L -1 compared to 44.4 g·L -1 The growth of the deletion mutant Δcgt was analyzed in several screening experiments in liquid culture using media in the range of 159 - 681 mOsmol·kg -1 No differences in the growth and survival rate of the deletion mutant Δcgt were observed compared to the wild type under all test conditions.

[0247] Surprisingly, under different pH and osmotic pressure conditions, no obvious physiological effects were observed due to the deletion of the cgt gene in the utilization of different carbon sources, either overly or limitedly. Therefore, the function of Cgt may only be revealed in its natural environment and in possible competition with other soil organisms. Interestingly, the inventors found similar independent specific CBM-20 domain proteins in 17 other prokaryotic species, most of which belong to the order Actinomycetales. Although rare, this shows at least a certain distribution and indicates that Cgt is not a strain-specific protein. Most of the species possessing 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 such as Cgt play extremely important functions in bacteria inhabiting this niche. The function of Cgt can be tested in the future by co-cultivation in direct contact with other microbial competitors.

[0248] Surprisingly, it was found that Cgt was unnecessary under the laboratory conditions tested, but the inventors observed a positive phenotype regarding acarbose production. Deletion of cgt achieved an increase in acarbose yield of 8.3 - 16.6%. The yield of the final product varied slightly between batch cultures, but the cgt mutants always functioned significantly better. This was shown over a period of several months in three independent shake flasks and several microscale cultures performed in maltose minimal medium (data not shown). Therefore, the improved production was consistent over a long period and in different culture environments.

[0249] This is due to the metabolic burden caused by the expression of the cgt gene in the wild type, assuming that it brings about a relaxation of energy and free resources in Δcgt. These resources are probably redirected towards the biosynthesis of acarbose, a growth-related product. No direct regulatory effect of cgt deletion on the expression of the acb gene was observed.

[0250] Analysis of the Functional Relevance of Carotenoid Formation Light-Dependent Carotenoid Formation and Oxidative Stress Decrease Acarbose Production in Actinoplanes sp. SE50 / 110 Actinoplanes is known to produce various soluble pigments, including the yellow, orange, and pink pigments of carotenoids (Parenti and Coronelli, 1979). The pigment of Actinoplanes sp. SE50 / 110 is orange. Its formation is enhanced when cultured under exposure to light. Since the pigment was also found in the supernatant, it seems to be soluble in an aqueous solution. After cell extraction and separation by thin-layer chromatography, spectral analysis showed absorption maxima at 450, 475, and 505 - 510, which was confirmed by absorbance scans performed during HPLC separation. Consistent with these findings shown by in silico reconstruction, Actinoplanes sp. SE50 / 110 has been shown to have a complete genetic apparatus for producing C40 - carotenoids similar to those of myxol from Salinospora tropica CNB - 440 (Richter et al., 2015; Wolf et al., 2017b) (Figure 21 and Table E12).

[0251] [Table 14] TIFF0007711052000059.tif233170TIFF0007711052000060.tif243170TIFF0007711052000061.tif56170

[0252] The genes for C40 - carotenoid biosynthesis are composed of three gene clusters: terpene clusters 1, 2a, and 2b (see Figure 21D).

[0253] In contrast to S. tropica, homologs of crtY and crtU, which encode cyclase and 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 study. They are located in terpene cluster 2b (Figure 21). CarR-domain cyclases are common in fungal, archaeal, and bacterial genomes (information obtained from NCBI's CDD search (Marchler-Bauer et al., 2017)). Since the pigment of SE50 / 110 is orange, terminal cyclization of the red precursor lycopene is likely and could be catalyzed by one or both of the CarR-domain cyclases. Similar to S. tropica, the carotenoid gene cluster of SE50 / 110 contains the glycosyltransferase CruC (Figure 21, Table E12). This strongly indicates glycosylated carotenoids, which, according to observations, seem to have polar characteristics as the pigment was found in the supernatant (Figure 22B).

[0254] Comparative genomic analysis by the software platform EDGAR 2.0 (Blom et al., 2016) showed similar terpene cluster arrangements in related species of Actinoplanes, but different organizations were found in Streptomyces (data not shown). This suggests that the gene arrangements found in SE50 / 110 and CNB-440 (Richter et al., 2015; Wolf et al., 2017b) are characteristic of the family Micromonosporaceae.

[0255] Furthermore, genes for the synthesis of the building blocks IPP and DMAPP via the MEP / DOXP-pathway (Table E12), the camphene-like monoterpene synthase (terpene cluster 3, Table E12), and a gene encoding a carotenoid cleavage dioxygenase (ACSP50_5522, Table E12) were found in the genome of SE50 / 110. The latter two may be involved in the formation of odorants (Yamada et al., 2015). The inventors observed that strong pigmentation was associated with production losses. This was confirmed by comparing the growth yields and acarbose yields of cultures exposed to light and cultures covered from light (Figure 22). Carotenoid production was induced, but the acarbose production and growth of the actinomycete species SE50 / 110 strongly decreased when exposed to the intensity of 22 - 44 μE (1 μmolE = μmol photons m -2 s -1 ) of globe light (36W, Osram 830U). Overall, a loss of 39% of the final acarbose concentration was monitored.

[0256] Deletion of merR in SE50 / 110 Induces Carotenoid Formation without Exposure to Light Since natural light or globe light was able to induce carotenoid formation (Figure 22B, C), this study searched for potential regulatory genes in SE50 / 110. A MerR regulator was found within terpene cluster 1 (ACSP50_0145, Figure 23).

[0257] The MerR-family mainly consists of activators that can respond to environmental stimuli such as oxidative stress, heavy metals or antibiotics (Brown et al., 2003). Indeed, some members of the MerR-family have been described as light-dependent activators or repressors of carotenoid biosynthesis in non-photosynthetic bacteria, for example, the related actinomycete S. coelicolor (Takano et al., 2005; Takano et al., 2006), the Gram-negative Thermus thermophiles HB27 (Takano et al., 2011) and the Gram-positive Bacillus megaterium QM B1551 (Takano et al., 2015) as both LitR. Here, cobalamin (vitamin B12) acts as a cofactor mediating photosensitivity as it can absorb ultraviolet and blue light: it can regulate the conformation and activity of the regulator by covalently binding to the regulator or by dropping off after photoexcitation (van der Horst et al., 2007). The regulatory mechanisms and binding sites are quite different: in T. thermophiles and B. megaterium, the promoter regions of LitR / crtB (Takano et al., 2011) or LitR and crtI (Takano et al., 2015) are repressed in the dark and relieved after irradiation, while litR in S. coelicolor seems to be an essential photoinducible transcriptional activator of the adjacent localized litS that encodes an ECF sigma factor and directs the transcription of carotenoid biosynthesis genes (Takano et al., 2005). Genes encoding ECF sigma factors do not occur within the gene cluster of SE50 / 110. In the Gram-negative bacterium Myxococcus xanthus, the B12-dependent MerR regulator is part of a complex regulatory cascade that includes eight additional regulator genes (Fontes et al., 2003; Galbis-Martinez et al., 2012 [here, the 'i' in 'Martinez' is correctly the letter i with an acute accent]).Indeed, no homologs of the M. xanthus - derived regulatory network were identified in the genome of SE50 / 110 by BLASTP - analysis (data not shown).

[0258] The MerR - family regulator ACSP50_0145 of Actinoplanes sp. SE50 / 110 contains an N - terminal HTH - motif and a C - terminal B12 - binding domain (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 (Perez - Rueda and Collado - Vides 2000 [here, the 'e' adjacent to the 'P' in 'Perez' is correctly the character with an acute accent on 'i']).

[0259] CRISPR / Cas9 deletion of the corresponding gene in SE50 / 110 strongly induced carotenoid formation without light exposure (Figure 24B, C). This confirms its function as a transcriptional repressor.

[0260] Indeed, it should be noted that the repressor / operator system is leaky, as the typical orange color is produced even in the wild - type without light exposure. According to this, the transcription of the genes crtEBI and idi (ACSP50_0146 - 0149) was only 2 - fold in ΔmerR compared to the wild - type under dark conditions (Figure 24E). These differences were significant for crtE, crtB, and idi. No effect on the transcription of the acb gene was observed.

[0261] However, in the context of this study, the question of whether pigment formation in ΔmerR affects the formation of micro - chemical acarbose was investigated. Again, higher carotenoid formation was associated with lower acarbose formation (Figure 24A, D). When irradiated, both the wild - type and ΔmerR were strongly pigmented, and the final acarbose concentration was similar for both strains, approximately 0.52 g·L -1was reached (Figures 24B, D). This corresponds to a decrease in acarbose production of approximately 38% compared to the wild type under dark conditions (0.83 g·L -1 is reached). This follows previous growth experiments of the wild type as described herein. Under dark conditions, ΔmerR produces approximately 15% less acarbose than the wild type (0.70 g·L -1 ) (Figure 24D). These production losses are suggested to be attributed to the waste of resources due to carotenoid formation in the deletion mutant (Figure 24C). As a conclusion, the production losses under light conditions (38 - 39%) may be due to additional photoinduced stress in both the deletion mutant and the wild type.

[0262] Performing a comparative transcriptome analysis of the wild type cultured under dark and light conditions using microarray technology shows a complex response at the transcript level affecting various genes (see Figure 25). Some of the differentially expressed genes show cellular responses to combat oxidative stress. Oxidative stress is caused by reactive oxygen species (ROS) formed by energy transfer (leading to singlet oxygen) or electron transfer (leading to superoxide, hydrogen peroxide, and hydroxyl radicals) (Ziegelhoffer and Donohue, 2009). At high concentrations, ROS are toxic and cause oxidation of proteins and membranes and DNA damage (Ziegelhoffer and Donohue, 2009; Gout, 2019).

[0263] In SE50 / 110, tyrosinase MelC (ACSP50_4950, formerly: ACPL_5017), a photoprotective agent involved in the formation of the brown pigment eumelanin (Wolf et al., 2016), and genes for riboflavin biosynthesis (ACSP50_6437-40) are transcribed more strongly when exposed to light (Figure 25). Riboflavin is a water-soluble photosensitizing oxidant that absorbs at 374 and 445 nm (Silva et al., 1999; Kim et al., 1993). It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These are cofactors of proteins involved in the redox metabolism of cells, light perception, DNA repair, and further functions (reviewed in Garcia-Angulo (2017) [where the 'i' in 'Garcia' should correctly be an 'i' with an acute accent]). Thereby, riboflavin and its derivatives are important micronutrients that enable cells to overcome oxidative stress (Chen et al., 2013).

[0264] Accordingly, several flavin-dependent oxygenases are also transcribed more strongly when exposed to light. One of them is annotated as taurine dioxygenase, and this substrate is a degradation product of cysteine. Sulfur-containing amino acids such as cysteine belong to the group of low molecular weight thiols (LMW thiols), can capture ROS, and can function as a redox buffer (Gout, 2019). Correspondingly, additional genes, probably involved in the metabolism and transport of cysteine and methionine, are transcribed more strongly in cells exposed to light.

[0265] Notably, several transcriptional regulator genes and the gene encoding sigma factor SigE (ACSP50_558) are also transcribed more strongly (Figure 25). SigE has been associated with the oxidative stress response in the photosynthetic bacterium Rhodococcus sphaeroides (reviewed in Ziegelhoffer and Donohue (2009)) and the envelope stress response in related species S. coelicolor (Hutchings et al., 2006) and C. glutamicum (Park et al., 2008). It is possible that SigE is involved in the oxidative stress response in SE50 / 110.

[0266] Interestingly, in the wild type exposed to light, the genes for carotenoid biosynthesis and the regulatory factor MerR are not significantly more strongly transcribed compared to the wild type hidden from light. This is worthy of note since a clear effect of light on carotenoid formation can be observed in the wild type. Carotenoid synthesis occurs both in the dark and in the light, and the enhancement of relative transcript levels is rather moderate in the regulatory factor mutants (see above), so the effect on transcript levels may not be prominent. For example, it is assumed that there may be further regulation of carotenoid synthesis at the protein level or metabolome level by the degradation of carotenoids or terpene precursors by carotenoid cleavage dioxygenase (ACSP50_5522). However, according to the results obtained from the wild type microarray, crt gene expression does not seem to be a major target of the overall oxidative stress response, similar to the findings from Rhodococcus sphaeroides (reviewed in Ziegelhoffer and Donohue (2009)).

[0267] In summary, illumination appears to induce an oxidative stress response and has a significant impact on the distribution of metabolic resources towards growth, carotenoid, and acarbose formation. The regulation of carotenoid biosynthesis is decoupled from the overall response to oxidative stress, and further research is needed. From the perspective of redirecting metabolic flux towards acarbose production, it is desirable to better understand these processes in the future. Since sigma factor SigE is more highly transcribed when exposed to light, it may be involved in the oxidative stress response.

[0268] Apart from light stress, this study demonstrates that a majority of the production loss can be directly allocated to carotenoid formation. Since carotenoids in non-photosynthetic bacteria have been shown to protect against photodynamic killing (Mathews and Sistrom, 1959), they are thought to function as photoprotectants (Lee and Schmidt-Dannert, 2002). Since the influence of light can be excluded by simple structural means, it is assumed that carotenoid formation is distributable under laboratory conditions. To improve acarbose production, the carotenoid biosynthesis pathway can be switched, for example, by deletion of the central gene crtI, and used in strain development. Since carotenoids affect membrane fluidity (Gruszecki and Strzałka, 2005 [here, the 'ł' in 'Strzałka' should be correctly stroked]), the lack of C40-carotenoids may also affect the surface layer and mycelial tissue of Actinoplanes sp. SE50 / 110. Regarding production, the degradation of mycelial clumps is advantageous for increasing the mycelial surface and the number of biochemically available cells.

[0269] Overexpression of acbB and gtaB The expression vector pSETT4 was tested for the acbB and gtaB genes. Both genes, acbB and gtaB, are likely involved in amino sugar synthesis, which is probably the 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 show an increase in protein amount in the cytosolic fraction of the acarbose-producing bacterium.

[0270] pSETT4gap and pSETT4tip Vectors for Overexpression of Single Genes A novel cloning system enabling easy cloning and overexpression of specific genes in Actinoplanes strains such as Actinoplanes sp. SE50 / 110 was implemented. For this purpose, a strong promoter of the gene gapDH from Eggerthella lenta was cloned in front of the lacZ-cassette of the pSET152-backbone. The gene lacZ is transcribed under the control of the lac-promoter and is adjacent to the recognition site of the restriction enzyme BsaI, enabling the replacement 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). Since strong expression requires strong termination, T4-terminators were introduced upstream and downstream of the cloning side of the novel expression system. The use of T4-terminators has already been successful in the pGUS-cloning system developed by Myronovskyi et al. (2011). Whole-track RNAseq analysis of the pGUS-integration mutants performed here showed that the T4-terminator efficiently blocks transcription and prevents read-through from the integrase gene to the gene of interest. As shown by preliminary experiments, the T4-terminator has no side effects on the transcription of the acb gene when introduced into Actinoplanes sp. SE50 / 110 via pSET152-integration.

[0271] Furthermore, sequencing of a concentrated primary transcript library derived from promoter-screening experiments identified two putative promoters behind the gene of interest in the antisense orientation (Figure 26). These two pseudo-promoters were removed in the new expression system to prevent antisense transcription. Furthermore, an additional (third) T4-terminator was introduced in the opposite orientation behind the cloning side to prevent additional putative antisense read-through.

[0272] To enable exchange of promoter sequences, NdeI and KpnI restriction sites were introduced. In this study, the strong gapDH-promoter was exchanged for the moderately strong tipA-promoter from S. lividans. This showed that this system can be easily modified, for example, to regulate it for other species of the order Actinomycetales. Vectors (named pSETT4gap and pSETT4tip) were tested for strong and moderately strong overexpression of the genes acbB and gtaB.

[0273] Moderate Overexpression of acbB Leads to Improved Acarbose Formation dTDP-D-glucose-4,6-dehydratase AcbB is thought to be involved in the production of the activated amino sugar from D-glucose-1P, the feeding pathway of acarbose biosynthesis (Figure 1): It was found that an increase in AcbB-activity improved the supply of modified precursors: Briefly, two overexpression variants were generated based on the expression vector pSETT4 described elsewhere in this specification. In these variants, acbB is transcribed under the control of a moderately strong tipA-promoter or a strong gapDH-promoter. As previously published (Schaffert et al., 2019), expression vectors using native promoters did not lead to significant overexpression of the genes of the Acb gene cluster. Therefore, native promoters were used in both the pSET152- and pSETT4-vector backgrounds as controls. Growth and acarbose formation were monitored in two shake flask cultures in maltose minimal medium (Figure 27).

[0274] The variant with acbB transcribed under the control of the heterologous tipA-promoter showed enhanced acarbose production compared to the control strain: the yield coefficients increased by 48.6 and 51.9% in two independent cultures compared to the empty vector control (Figure 28). The use of the strong gapDH-promoter led to a slight increase in the acarbose yield coefficient (Figure 28).

[0275] 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 (Figure 29). Thus, the supply of the activated glucose moiety seems to be ensured. In this variant, an increase in the mass M / z = 545 [M-H + was found (Figure 29, approximately 41%). Without being bound by theory, this intermediate accumulates, for example using pSETT4tip::acbB, by moderate AcbB-overexpression.

[0276] Enhanced expression of acbB was observed at the start of the growth phase within the expected range: the most potent overexpression was achieved by using the gapDH-promoter (log2(fold change) = 6.54), followed by the tipA-promoter (log2(fold change) = 4.06) (Figure 30). Use of the native promoter did not result in a significant increase in the relative transcription of acbB. This was tested in both the pSET152-vector background and the pSETT4-vector background (Figure 30). As shown for acbA and acbV, there was no significant effect on further genes of the acb gene cluster (Figure 30). However, the transcription of acbA in pSETT4tip::acbB (log2(fold change) = 1.87) was slightly higher.

[0277] Notably, the transcription profile during the linear growth phase differed from that of the initial growth phase: here, only a doubling of transcription was achieved by using the gapDH-promoter (log2(fold change) = 2.05), while by using the tipA-promoter, overexpression of acbB was maintained but to a lesser extent (log2(fold change) = 3.33) (Figure 30, Figure 31).

[0278] In overexpression mutants containing heterologous promoters, the relative transcription of acbB decreased from 4.06-fold to 3.33-fold (log2(fold change)) between the two sampling times in pSETT4tip::acbB, and from 6.54-fold to 2.05-fold in pSETT4gap::acbB. It is thought that the transcription of the chromosomal acbB copy is downregulated in these mutants, while the transcription of the vector copy is maintained by the heterologous promoter. The difference in acbB-transcription at different sampling times further suggests that the downregulation of acb gene transcription occurs earlier and more strongly in pSETT4gap::acbB compared to pSETT4tip::acbB. The overexpression of acbB (pSETT4gap::acbB and pSETT4tip::acbB) appears to decelerate during the linear growth phase.

[0279] In summary, while moderate overexpression of acbB, particularly by using the tipA-promoter, seems to be beneficial for acarbose production, strong overexpression by using the gapDH-promoter seems to have a smaller effect on acarbose formation. Further improvement of acarbose formation can be achieved by changing the expression level of acbB, for example, by using a selective promoter from promoter screening, or by introducing multiple gene copies.

[0280] In summary, this study demonstrates that moderate overexpression of AcbB increases the acarbose yield, probably due to an improved amino sugar supply. A positive effect on acarbose production was observed by moderate overexpression of acbB (e.g., by using the tipA-promoter), and more than about 50% acarbose was obtained in two independent cultures. Thus, improvement of acarbose biosynthesis by overexpression of the specific acb gene was achieved.

[0281] Moderate Overexpression of gtaB Leads to Improved Acarbose Formation GtaB is thought to catalyze the interconversion of UDP-glucose and glucose-1P. Surprisingly, overexpression of GtaB was found to induce acarbose formation. Without being bound by theory, this could occur by improved deployment of the precursor glucose-1P. As shown by shake flask culture in minimal maltose medium (Figure 32), the final acarbose yield coefficient of the overexpression mutant of gtaB introduced into pSETT4tip increases to 8.56%. Interestingly, acarbose formation increases particularly from the late logarithmic to the stationary phase. In the overexpression mutant, the relative transcript level of the gene gtaB increased 2.64-fold (log2(fold change)) (Figure 33). Activated sugar metabolism is not thought to be stored because it is linked to or redirected to other metabolic pathways. However, as shown in previous experiments, supply can be severely disrupted. Analysis of the intracellular metabolome shows that the amounts of phosphorylated hexoses and / or UDP-glucose are comparable (Figure 34). Therefore, the pool of activated C6-sugars is not significantly affected by overexpression of gtaB.

[0282] Interestingly, a significant decrease in mass M / z = 545 [M-H + was found in pSETT4tip::gtaB (a decrease of approximately 48%), which could correspond to dTDP-4-keto-6-deoxy-D-glucose, the proposed product of AcbB. This could indicate that the flow through the synthetic pathway is more balanced as the accumulation of this metabolite is decreased compared to the empty vector control and the AcbB overexpression mutant (Figure 34). In summary, the introduction of a second gene copy of gtaB has an as yet unclear effect on the distribution of cellular goods but has a positive effect on acarbose production. Transferring this construct to the actinoplanes production strain may increase the beneficial effect because here the demand for precursors is higher compared to the wild type. Strong overexpression of AcbB results in an imbalance in the overexpression of acbB and gtaB complexed in glucose-phosphate metabolism and will probably further improve acarbose production beyond the effects observed for single overexpression.

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Claims

1. A method of manipulating an Actinoplanes strain for improved production of acarbose, the method comprising manipulating the Actinoplanes strain such that (i) the expression of the extracellular small carbohydrate-binding protein Cgt having the amino acid sequence represented by SEQ ID NO: 20 is lost or attenuated . The method as described above.

2. The method according to claim 1, wherein the method comprises (i) deletion or mutation of the gene encoding the extracellular small carbohydrate-binding protein Cgt having the amino acid sequence represented by SEQ ID NO: 20 . The method as described above.

3. An Actinoplanes strain for producing acarbose, wherein the Actinoplanes strain has been genetically engineered for loss or attenuated expression of the extracellular small carbohydrate-binding protein Cgt having the amino acid sequence represented by SEQ ID NO:

20. The Actinoplanes strain as described above.

4. The Actinoplanes strain according to claim 3, wherein the Actinoplanes strain is a cgt deletion mutant.

5. An Actinoplanes strain for producing acarbose according to any one of claims 3 to 4, wherein the strain contains a vector, and the vector contains an expression cassette for AcbB having the amino acid sequence represented by SEQ ID NO: 13 and / or an expression cassette for GtaB having the amino acid sequence represented by SEQ ID NO: 19, and / or an expression cassette for MerR having the amino acid sequence represented by SEQ ID NO:

22. The Actinoplanes strain as described above.

6. Use of the Actinoplanes strain according to any one of claims 3 to 5 in the production of acarbose.

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