Method for improving the formation of acarbose

Abstract

The present invention relates to actinomycetes strains for improving acarbose formation. The present invention provides Actinomycetes strains engineered to overexpress dTDP-D-glucose-4,6-dehydratase (AcbB) and / or uridyltransferase (GtaB). Also provided are Actinomycetes strains engineered to have reduced or absent expression of small carbohydrate binding protein (Cgt) and / or reduced or absent expression of genes necessary for carotenoid synthesis. Also provided are tools, methods and means for generating these strains.

Description

Field of the Invention

[0001] The present invention relates to Actinomycetales strains for improving acarbose formation. The present invention provides Actinomycetales strains engineered to overexpress dTDP-D-glucose-4,6-dehydratase (AcbB) and / or uridyltransferase (GtaB). Also provided are Actinomycetales strains engineered to have reduced or absent expression of small carbohydrate binding protein (Cgt) and / or reduced or absent expression of genes necessary for carotenoid synthesis. Also provided are tools, methods and means for producing these strains. Background Art

[0002] Acarbose

[0003] The therapeutic agent acarviosyl-maltose (acarbose) has been used in the medical treatment of diabetes since 1990 (Wehmeier and Piepersberg 2004; Wehmeier 2004). It will support the patient's strict dietary plan and prevent sugar peaks when consuming a high-carbohydrate meal. After oral administration, acarbose inhibits intestinal α-glucosidase, which leads to a delayed release of monosaccharides from a diet containing starch and sucrose. Thereby, acarbose helps control the rate of absorption of monosaccharides into the blood system and leads to a reduction in postprandial blood and serum sugar levels, which is considered to be crucial in the context of cardiovascular disease mortality.

[0004] Industrial Applicability

[0005] Acarbose is known and is marketed as Glucobay (Bayer AG) in Europe and China, as Precose (Bayer Pharmaceuticals) in North America, and as Prandase (Bayer AG) in Canada. As an important and highly desired drug for the treatment of diabetes, acarbose needs to be provided in high yield and quality. As the incidence of type II diabetes continues to increase worldwide, optimization of product yield and quality is a current concern.

[0006] Acarbose-producing strains

[0007] Acarbose is naturally produced by different actinomycetes, such as Streptomyces coelicoflavus ZG0656 (Geng et al. 2009), Streptomyces glaucescens GLA.O (Rockser and Wehmeier 2009; Ortseifen et al. 2015) and Actinomyces aeruginosa SE50 / 110 (reviewed by Wehmeier and Piepersberg 2004), the latter of which is a wild-type industrial production strain (Ortseifen 2016; Mahmud et al. 1999). The genus Actinomyces aeruginosa was first introduced by Couch (1950) as a member of the phylum Actinomycetes, order Actinomycetales, family Micromonosporaceae. Actinomycetes SE50 / 110 (ATCC 31044, CBS 674.73) is a slow-growing natural derivative of SE 50 (ATCC 31042, CBS 961.70) (Frommer et al., 1973). SE50 was isolated from soil samples near coffee plantations in Kenya during a screening project at Bayer AG in 1970 (Frommer et al., 1972). When maltose is provided in the culture medium, SE50 / 110 produces about 1 g·L -1 Acarbose (Wendler et al., 2014). Other production strains have been engineered, as described in, for example, (EP2601209B1) and (CN103298828B).

[0008] For A. aeruginosa SE50 / 110, it was shown that the biosynthesis of akavier-sugar is dependent on the supply of carbon source in the medium (Wendler et al. 2014). When grown on glucose, akavier-glucose is formed as the main compound, while when grown on maltose, akavier-maltose is mainly formed (Wendler et al. 2014), and when grown on maltotriose, akavier-maltotriose is mainly formed (Ortseifen 2016).

[0009] Due to its medical and industrial relevance to the wild-type industrial acarbose production strain, A. aeroplanum SE50 / 110 has been extensively studied in the last years: the complete genome (Schwientek et al. 2012), transcriptome (Schwientek et al. 2013) and proteome (Wendler et al. 2015b; Wendler et al. 2015a; Wendler et al. 2013) were comprehensively analyzed. In 2017, this resulted in an optimized genome sequence and improved annotation (GenBank: LT827010.1) (Wolf et al. 2017b). In addition, an intergeneric conjugation system (Gren et al. 2016) as well as advanced genome editing tools by using CRISPR / Cas9 (Wolf et al. 2016) were established, allowing targeted genetic engineering. However, for A. aeroplanum SE50 / 110, a reliable expression system that enables moderate to strong gene expression is lacking. Since no suitable system for moderately strong overexpression of single genes existed previously, different strategies were tested and evaluated according to the present invention, which led to the development of a new expression system called pSETT4.

[0010] Biosynthesis of Acarbose

[0011] The biosynthetic pathway of acarbose is based on a monofunctional enzyme that catalyzes a single step ( Figure 1)(Wehmeier and Piepersberg 2009). According to the model of Zhang et al. (2002), the biosynthesis proceeds via the intermediate valienone-7P. In the refined optimization of Wehmeier (2003), the reduction and dehydration steps were changed, leading to valiolol-7P as an intermediate. Since the order of the steps is unknown, they are shown in brackets. The first step of acarbose biosynthesis, the cyclization reaction of AcbC from sedo-heptulose-7P to form 2-epi-5-epi-valiolone, is missing in this illustration. Although the acarbose biosynthetic pathway has become the focus of research in the past few decades, the biosynthetic pathway has not yet been fully elucidated. Only the first three steps of the biosynthesis have been experimentally confirmed. AcbC (ACSP50-3607), the first enzyme in acarbose biosynthesis, catalyzes the cyclization reaction from sedum-heptulose 7P7P to 2-epi-5-epi-valiolone (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 isomerized to 5-epi-valiolone-7P by the cofactor-independent epimerase AcbO (ACSP50-3606) (Zhang et al., 2002; Zhang et al., 2003).

[0012] The remaining steps of the model are based on protein homology and functional predictions (Zhang et al. (2002), Wehmeier (2003), Wehmeier and Piepersberg (2004), Wehmeier and Piepersberg (2009) and Wendler et al. (2013)): NADH-dependent (polyol) dehydrogenase / reductase AcbL (ACSP50_3604) and cyclitol dehydrogenase / oxidoreductase AcbN (ACSP50_3605) have been suggested to catalyze the reduction and 5,6-dehydration to 1-epi-valienol-7P. Phosphorylation to 1,7-diphospho-1-epi-valienol is thought to be catalyzed by 1-epi-valienol-7-phospho-1-kinase AcbU (ACSP50_3595) and / or hydrolase AcbJ (ACSP50_3600). Nucleotidylation to NDP-1-epi-valienol-7P is probably catalyzed by the GlgC-related NDP-polyol synthase AcbR (ACSP50_3597) (1-epi-valienol-1,7-diphosphate-adenylyltransferase), and transfer of the activated intermediate to the activated amino sugar appears to be mediated by the glycosyltransferases AcbI (ACSP50_3599) and / or AcbS (ACSP50_3596) to generate acarviosine-7P. The activated amino sugar is thought to be synthesized from D-glucose-1-phosphate in three steps (Wehmeier and Piepersberg 2004; Wehmeier and Piepersberg 2009; Zhang et al. 2002), which are: (i) nucleotidylation to 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).

[0013] Glucose-1P is a branched metabolite that exhibits important roles in different pathways, such as glycogen metabolism, galactose metabolism, and glycolysis after conversion to glucose-6P (Frey 1996; Purves 2006). UDP-glucose-1P uridyltransferase GtaB catalyzes the interconversion of glucose-1P and UDP-glucose.

[0014] Finally, maltose may be transferred in a one-step reaction by AcbS (Hemker et al., 2001). However, it has also been proposed that AcbI or AcbJ catalyze the transfer reaction (Wehmeier and Piepersberg 2004; Wendler et al. 2013). Another candidate for this reaction could be the amylomaltase AcbQ (ACSP50_3601).

[0015] In Actinomycetes SE50 / 110, the biosynthetic genes are organized in the acarbose biosynthetic gene cluster (acb gene cluster), which was first identified by Stratmann et al. in 1999 and subsequently sequenced (GenBank: Y18523.4) (Stratmann et al. 1999; Thomas 2001). This cluster contains 22 genes ( Figure 2 ).

[0016] In addition to the already mentioned biosynthetic genes (acbCMOLNUJRSIVBA), this cluster encodes functions in extracellular starch degradation (AcbEZ, ACSP50_3610 and ACSP50_3590), transglycosylation (AcbD, ACSP50_3611) and acarbose export (AcbWXY, ACSP50_3591-3). In addition, acarbose-7-kinase (AcbK, ACSP50_3602) and an 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.

[0017] Actinomycete proteins of possible metabolic relevance

[0018] The single CBM-20 domain protein Cgt is one of the most strongly expressed genes in A. acarbose SE50 / 110 and derived acarbose-producing strains (Ortseifen 2016; Wendler et al. 2015a; Schwientek et al. 2013). It is secreted via the Sec pathway and represents 8% of the total secretory proteome of this organism according to SignalP analysis (Almagro Armenteros et al., 2019) (data not shown). Cgt contains a CBM-20 domain of 149 amino acids and fold family 1 functional group A, characterized by a β-sandwich structure (Schwientek et al., 2013; Guillén et al., 2010). Members of this family have been described to bind starch (Guillén et al. 2010).

[0019] Methods for gene deletion in actinomycetes

[0020] The establishment of the intergeneric ligation system (Gren et al. 2016) and CRISPR / Cas9 technology (Wolf et al. 2016) allowed genome editing in Actinoplanes SE50 / 110.

[0021] In addition, according to the present invention, the inventors successfully established a new deletion system by homologous recombination, which uses an integrase-free vector backbone and CodA for counter-selection, as described in Zhao et al. (2017). As a result, the genetic toolbox of Actinoplanes SE50 / 110 can be further expanded. As a proof of principle, the deletion of the exemplary gene cgt of the new deletion system was successfully tested. Homologous recombination (HR) is a common process in actinomycetes, which can be technically used to generate deletion mutants by double exchange. Temperature-sensitive replicons, such as the PSG5 replicon, can support and promote this process (Du et al. 2015; Garg and Parry 2010; Myronovskyy et al. 2009; Zhang and Parry 2007). Other methods exist in the art, such as CRISPR-base editing systems for single nucleotide exchange, CRISPR-BEST according to Tong et al. 2019, CRISPRi / dCas9 according to Qi et al. 2013, RNA interference, etc.

[0022] Methods for Gene Overexpression in Actinomycetes

[0023] Actinomycetes SE50 / 110 has been extensively studied over the past few decades. Suitable expression systems are difficult to design, see Schaffert et al. (2019). The entire contents of this publication, especially the description of expression systems and promoters for genetic manipulation of actinomycetes, are included in this article in their entirety.

[0024] Previous studies have shown that genes were successfully expressed using pKC1139 in A.teichomyceticus (Horbal et al. 2012). However, the replicative pSG5-based vector pKC1139 (constructed by Bierman et al. (1992)) proved to be unsuitable for expressing homologous genes in A.teichomyceticus SE50 / 110 because unwanted vector integration occurred by homologous recombination, see Schaffert et al. (2019). This seems to be a favorable process, presumably due to the high metabolic cost of vector replication. Without being bound by theory, the protein encoded by ACSP50_7170 in SE50 / 110 (predicted to be recombinase A (RecA)) may catalyze the recombination process. Interestingly, no homologs of recA were found in the genome of A.teichomyceticus. The presence of RecA in A. planarians SE50 / 110 and its absence in A. teicoplanin provide a conclusive explanation why HR-mediated vector integration has not been reported previously for A. teicoplanin. Thus, by deleting the recombinase gene recA in A. planarians SE50 / 110, a pSG5-based replication-competent expression system can be achieved.

[0025] Other replicative Streptomyces-E. coli shuttle plasmids, such as pKC1218 based on the SCP2*-replicon (Kieser et al., 2000) and pSOK101 based on the pIJ101-replicon (Zotchev et al., 2000), do not produce conjugative bodies with A. aeruginosa SE50 / 110 (Gren 2017). These replicons may be unstable or inactive in SE50 / 110, which is consistent with the findings of the related species A. aeruginosa (Horbal et al. 2012).

[0026] By using an integrative vector system, genetic replication can be achieved by the integration of a complete vector carrying additional gene copies at different genomic locations. This process is mediated by a bacteriophage integrase. The bacteriophage integrase catalyzes the targeted and unidirectional recombination of two attachment sites: attP on the plasmid and attB in the host chromosome (te Poele et al., 2008). After integration, the vector flanks the left attachment site (attL) and the right attachment site (attR), which is derived from attP-attB-recombination (te Poele et al., 2008).

[0027] Four different integration vectors have been described for A. aeruginosa SE50 / 110 (Gren et al. 2016): two are based on bacteriophage Integration mechanism (pSET152 and pIJ6902). Vectors pRT801 / 2 and pSOK804 are based on phage and VWB-phage integration mechanisms. However, by using the native promoter, no doubling of the relative transcript amounts was achieved, see Schaffert et al. (2019).

[0028] Evaluation of homologous and heterologous promoters for integrating vectors

[0029] Schaffert et al. (2019) provides a method for evaluating homologous and heterologous promoters in terms of strength, which is incorporated herein in its entirety. -based vector pSET152 was used for promoter screening in Actinoplanes SE50 / 110 (Gren et al. 2016). The promoter strength of 13 homologous and heterologous promoters was analyzed at the protein level, and 12 of them were analyzed at the transcript level (Table 1, Figure 3 ).

[0030] Table 1. Constructs with the reporter gene gusA tested in promoter screening experiments

[0031]

[0032]

[0033] Strategy

[0034] For the present invention, acarbose metabolism was investigated by gene deletion and overexpression, resulting in a set of tools and methods to engineer strains for improved acarbose production. To improve acarviose synthesis, three different strategies were followed: (i) increasing the gene dosage of the acb gene to enhance the flux through acarbose biosynthesis, (ii) configuring precursors for acarbose biosynthesis, and (iii) reducing the metabolic load ( Figure 4 ). The pathways of each of these related strategies surprisingly led to improved acarbose formation: by overexpressing the dTDP-D-glucose-4,6-dehydratase AcbB, the final acarbose concentration was significantly increased by about 50%. By overexpressing the uridyltransferase GtaB, the acarbose yield was increased by 8.5%, presumably because of the improved supply of the precursor glucose-1P. By functional deletion of the small carbohydrate binding protein Cgt, acarbose formation was significantly enhanced by 8-16%, probably because of the reduced metabolic load thereby. This enhancement was robust over long periods of time and in different culture configurations.

[0035] Furthermore, growth experiments of the wild type and the regulator mutant ΔmerR exposed to light and protected from light were performed, revealing the negative effects of light-induced stress and carotenoid formation on acarbose production. Therefore, acarbose production could be further improved by reducing carotenoid formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 . Model for the biosynthesis of acarbose from 2-epi-5-epi-valiolone in Actinomycetes SE50 / 110. Eleven steps are shown for the biosynthesis of acarbose from 2-epi-5-epi-valiolone (Zhang (2002)).

[0038] Figure 2 .Acarbose biosynthesis gene cluster and gene distribution in the Actinomycetes SE50 / 110 genome (GenBank: LT827010.1) (Schaffert, et al. 2019).

[0039] Figure 3 .Promoter screening at the protein and transcript levels in the A. aeruginosa SE50 / 110 strain, see Table 1. The normalized glucuronidase activity (absolute value) is shown on the left, and the relative transcript amount of the gusA gene calculated by RT-qPCR is shown on the right. For the glucuronidase assay, the slope of the indigo absorption curve was calculated by linear regression and normalized to the dry cell weight. The significance of the normalized activity compared to PGUs was tested in a two-sided t-test (p-value: P 2475 :0.8889,P efp:3.048e-07,P cdaR :8.967e-07,P rpsL :1.296e-08,P rpsJ :0.0003677,P cgt :2.183e-06,P tipA :0.0001651,P apm :0.0001078,P ermE* :0.007406,P katE :0.002577,P moeE5 :0.001809,P gapDH :0.0005821,P act The relative transcript amount of the gusA gene was analyzed relative to the pGUS-vector (set to 1). For the act-promoter, RNA could not be isolated due to a severe growth defect. For the residual promoter, a significant increase in the relative transcript amount was measured (p value of the two-sided t-test: P 2475 :0.0001133,P efp :4.871e-05,P cdaR :0.002509,P rpsL :9.928e-06,P rpsJ :1.167e-08,P cgt :5.911e-08,P tipA :7.158e-06,P apm :4.596e-05,P ermE* :0.0009364,P katE :0.0001373,P moeE5 :0.0002518,P gapDH :4.207e-06). The significance levels of the calculated p-values ​​are shown by asterisks: *<α=5%,**<α=1%,***<α=0.1%. The figure is published in (Schaffert, et al. 2019).

[0040] Figure 4 . Strategies for improving acarbose production. Three different strategies are provided to improve acarbose production: 1. increasing the gene dosage of acarbose biosynthetic genes, 2. the distribution of acarbose biosynthetic precursors, and 3. reducing the metabolic load by gene deletion. The target genes evaluated in this study are shown. Further, an overexpression system must be implemented to overexpress single genes.

[0041] Figure 5.Chemical structure of acarbose. Acarbose is a cyclitol-containing aminoglycoside composed of a pseudodisaccharide (valienaminyl-4-amino-4,6-dideoxyglucose) (called acarviose) and maltose. The two are linked by an α-1,4-glycosidic bond. Figure published in Wolf 2017.

[0042] Figure 6 .Vector card of the new cloning system pSETT4 (see SEQ ID NO.110, SEQ ID NO.111). A promoter (such as the strong promoter of the gene gapDH from Eggertella tarda or the tipA promoter) is cloned in front of an expression cassette, such as a lacZ-cassette. The lacZ-cassette is flanked by recognition sites for restriction enzymes, such as BsaI. The restriction sites enable the exchange of lacZ by the gene of interest by Gibson assembly, restriction / ligation cloning or Golden Gate cloning. For termination, T4 terminators are introduced before and after the cloning site. After the cloning site, two antiparallel oriented T4 terminators will prevent read-through from both directions. In order to exchange promoter sequences, other restriction sites are introduced, such as NdeI and KpnI restriction sites. In addition, the vector contains the integrase gene int and the bacteriophage The pUC1 / pMB1 / pBR322 / pUC locus contains the junction site attP, the transfer origin (ncP) and relaxosome gene traJ, the high copy number ColE1 / pMB1 / pBR322 / pUC replication origin and the resistance gene (here: the apramycin resistance gene aac(3)IV (apmR)).

[0043] Figure 7 . Scheme of the new deletion system and the process during homologous recombination (first and second crossover). R or R Selection for vector integration was performed by using 5-fluorouracil for counter-selection (secondary crossover, codA-mediated sensitivity).

[0044] Figure 8 .Workflow of the novel deletion system using homologous recombination.

[0045] Fig. 9BlastP analysis of the amino acid sequence of .Cgt resulted in the identification of 17 other proteins consisting of a single CBM-20 domain. A protein tree was created and visualized based on a multiple sequence alignment (Altschul et al. 1990) performed by BlastP. The protein tree shows the distances of the 18 single CBM-20 domain proteins and their hosts identified by NCBI accession numbers. In brackets, the sequence identities and positives of the BlastP analysis are shown in percentages.

[0046] Fig.10 Growth of wild-type A. aeruginosa SE50 / 110 in basal medium supplemented with different carbon sources (equal C-molar amounts). Cell dry weights and standard deviations (n glc =3,n mal =5,n cel =4,n lac =3,n ara =5,n starch =5).

[0047] Fig.11 .A. Relative transcript amounts of cgt in A. aeruginosa SE50 / 110 grown on basal medium supplemented with starch, C-Pur, glucose, galactose, cellobiose or lactose as carbon sources compared to cultures grown on maltose basal medium. Difference testing in a two-sided t-test showed significant differential gene expression of the cgt gene on carbon sources glucose (p value = 0.002848), galactose (p value = 0.002945) and lactose (p value = 0.00114) compared to maltose. B. Relative transcript amounts of cgt in A. aeruginosa SE50 / 110 grown on basal medium supplemented with starch, C-Pur, glucose, galactose, cellobiose or lactose as carbon sources compared to cultures grown on maltose basal medium. -1 Compared with the cultures grown on maltose, the cultures grown on maltose supplemented with 44.40 g·L -1 Relative transcript levels of cgt in A. aeruginosa SE50 / 110 grown on maltose-based medium containing maltose. The difference test in a two-sided t-test showed that the gene expression of cgt was significantly reduced in the medium containing reduced amounts of maltose (p value = 0.04141).

[0048] Fig.12 Growth of wild type and deletion mutant Δcgt of A. aeruginosa SE50 / 110 in basal medium supplemented with different carbon sources. Cell dry weight and standard deviation are shown over time (wild type: n glc =3,n mal =5,n cel =4,n lac =3,n ara =5,n starch =5,Δcgt:n glc =2,n mal=5,n cel =4,n lac =4,n ara =5,n starch =5).

[0049] Fig.13 Final dry cell weights obtained from growing the wild type and Δcgt mutant in basal medium supplemented with six different carbon sources. Error bars indicate standard deviation.

[0050] Fig.14 Growth of Δcgt and wild type under limiting amount of starch as carbon source. The medium was supplemented with 1 g·L -1 , 2g·L -1 , 3g·L -1 , 4g·L -1 and 5g·L -1 starch, and in m2p labs The backscatter signal and the standard deviation of at least three biological replicates are shown in the bar graph. No Δcgt-induced growth limitation was observed. -1 , and even found that growth was significantly enhanced (p value of two-sided t test: 0.006141, n wt =3,n Δcgt =4).

[0051] Fig.15 Final dry cell weight for pH screening experiment in maltose basal medium. Wild type and Δcgt mutant of Actinomycetes SE50 / 110 were obtained from m2p-Labs The cells were grown in 48-well FlowerPlates in the system in a 1 mL reaction volume. No significant differences in final dry cell weight were observed over the pH range of 4 to 7 (tested by a two-sided t-test, n wt =3,n Δcgt =4).

[0052] Fig.16 .m2p-labs Osmotic pressure tolerance screening in the system: Final cell dry weight in maltose basal medium with maltose monohydrate concentrations between 3.6 and 108.1 g L -1 No significant growth differences were observed (tested by two-sided t test, n wt =3,n Δcgt =4).

[0053] Fig.17 .m2p-Labs Osmotic pressure tolerance screening in the system: Final cell dry weight of osmotic pressure screening experiment in maltose basal medium. Different osmotic pressures were obtained by adding inositol at concentrations ranging from 0 mM to 280 mM. No significant growth difference between wild type and Δcgt was observed (tested by two-sided t test, n wt =3,n Δcgt =4).

[0054] Fig.18 The wild type and Δcgt mutant of Actinomycetes SE50 / 110 were supplemented with 11.0 g·L -1 Maltose and 10.0 g·L -1 Growth and acarbose production in NBS complex medium with glucose monohydrate. No differential growth was detected. During the growth phase, acarbose concentration was significantly increased as measured in Δcgt (significance of t-test after 49 hours of culture: p value = 0.006778, n wt-acb =3,n Δcgt-acb =3,n wt-cdwGlc =4,n Δcgt-cdwGlc =3,n wt-cdwMal =4,n Δcgt-cdwMal =4).

[0055] Fig.19 A. Final yield coefficient of acarbose relative to cell dry weight in the bar graph. Error bars were calculated by Gaussian error propagation. B. Cell dry weight and acarbose concentration in the supernatant during cultivation in maltose-based medium (n cdw =5,n acb =4).

[0056] Fig. 20 Relative transcript amounts of genes acbZ, acbW, acbV, acbA, acbB, acbE and acbD of mutant Δcgt compared with wild-type A. aeruginosa SE50 / 110 grown on maltose-basal medium (n=3-6).

[0057] Figure 21. Reconstruction of carotene formation in A. planarian SE50 / 110. Putative homologous genes in A. planarian SE50 / 110 identified by BLASTX analysis against the NCBI database are shown. Reconstruction was performed with the aid of Kyoto Encyclopedia of Genes and Genomes (Kanehisa et al. (2014)).

[0058] A. The methylerythritol phosphate (MEP) pathway, also known as an alternative metabolic pathway to the mevalonate pathway, for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

[0059] BC: Carotene formation. B. Lycopene formation from isoprenoid precursors. C. Synthesis of the glycosylated carotenoid sioxanthin in Salinospora tropica CNB-440 (Richter et al. (2015) Figure 1 ).

[0060] D. Genomic organization of the genes identified in A. aeruginosa SE50 / 110. Gene cluster 2b showed homology to the sioxanthin gene cluster from Halospora tropicalis CNB-440 according to analysis by antiSMASH, a rapid genome-wide identification tool for annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genomes (Weber et al., 2015).

[0061] Fig. 22 . Growth, acarbose and pigment formation of Actinomycetes SE50 / 110 exposed to light or protected from light. A. Growth, acarbose and pigment formation of Actinomycetes SE50 / 110 exposed to light or protected from light (22-44 μE, 1 μE = μmol 光子 m -2 s -1 A. Cultivation of wild-type Actinomycetes SE50 / 110 in maltose-based medium with 5% paraformaldehyde. A. Cell dry weight of five biological replicates and acarbose concentration in the supernatant of three biological replicates are shown. B. Pellet and supernatant at the final incubation time. C. Growth and pigmentation in solid cultures on SFM agar plates exposed to or shielded from natural light.

[0062] Fig.23 . Location of genes encoding MerR-regulators in terpene cluster 1 and their distribution in the genome of Actinomycetes SE50 / 110 (see Figure 21 and Table E12). The genes of the cluster encode a MerR-like transcriptional regulator (ACSP50_0145), an isopentenyl-diphosphate delta-isomerase (idi, ACSP50_0146), a phytoene dehydrogenase (crtI, ACSP50_0147), a polyisoprenyl synthase (crtE, ACSP50_0148), a phytoene synthase (crtB, ACSP50_0149), a deoxyribodipyrimidine photolyase (ACSP50_0150), and a pyridine nucleotide-disulfide oxidoreductase (ACSP50_0151).

[0063] Figure 24. Growth, acarbose and pigment formation of Actinomycetes SE50 / 110 and deletion mutant ΔmerR exposed to light or protected from light. A. Growth, acarbose and pigment formation of Actinomycetes SE50 / 110 and deletion mutant ΔmerR exposed to light or protected from light (22-44 μE, 1 μE = μmol光子 m -2 s -1 ) in maltose-based medium. A. Cell dry weight of at least four biological replicates and acarbose concentration in the supernatant of three biological replicates are shown. B. Pellet and supernatant at the final culture time. C. Growth and pigment formation on solid medium (SFM agar plates). D. Maximum acarbose concentration (p-values ​​of two-sided t-test: wt dark vs. wt light: 0.003975, wt dark vs. ΔmerR dark: 0.09711, wt dark vs. ΔmerR light: 0.007043, ΔmerR dark vs. wt light: 0.02081, ΔmerR dark vs. ΔmerR light: 0.0002131). E. Relative transcript amounts of genes crtE (ACSP50_0148), crtB (ACSP50_0149), crtI (ACSP50_0147), idi (ACSP50_0146), and merR (ACSP50_0145) in deletion mutants compared to wild type (set to value 1) when cultured in the dark (p-values ​​of two-sided t-test: crtE: 0.04245, crtB: 0.01017, crtI: 0.07162, idi: 0.004366). Asterisks indicate significance levels: *p-value <α = 5%, **p-value <α = 1%, ***p-value <α = 0.1%.

[0064] Fig.25 Ratio / intensity plot of differentially transcribed genes in A. aeruginosa SE50 / 110 exposed to light compared to cultures grown in the dark. Ratios (log2(fold change)) were plotted relative to the mean intensity of the microarray experiment. Darker dots represent genes with significantly differential transcribed levels in cultures exposed to light compared to cultures protected from light.

[0065] Fig.26.ReadXplorer (Hilker et al., 2014) view showing the TSS of the putative antisense promoter behind the gene of interest in the pSET152-vector system. The TSSs were determined by sequencing the pooled primary transcript library. An exemplary stacked read of the integration vector-mutant mapped to pGUS:pAPM:gusA is shown. Two TSSs (enclosed by boxes) are located behind the gene of interest in the antisense orientation (A). These TSSs can be assigned to sequence motifs on the vector backbone (B), which are putatively recognized as promoter sequences by the σA / RNA polymerase complex. The conserved nucleotides of the -10- and -35-hexamers are highlighted. If present, the TG-dimer is shown in bold black letters. The distance between the hexamers is shown by s1; the distance between the -10 motif and the TSS is shown by s2.

[0066] Fig. 27 Growth and acarbose production of acbB overexpressing strains in maltose-basic medium. Two independent cultures are shown (A and B). The sampling times for RNA-isolation are indicated by t1 ("early growth phase") and t2 ("linear growth phase").

[0067] Fig.28 .Yield coefficients of acbB overexpressing mutants in maltose basal medium. Mutants with acbB transcribed under the control of the heterologous tipA-promoter showed an enhanced yield coefficient (about 50%), while only minor differences were observed for constructs with the gapDH-promoter. Errors were calculated by Gaussian error propagation. The significance of all differences was tested by two-sided t-test (abbreviations specified in the figures). Asterisks indicate the significance level: *p-value <α=5%, **p-value <α=1%, ***p-value <α=0.1%.

[0068] Fig.29 LC-MS analysis of intracellular metabolites of acbB-overexpressing mutants. The mass m / z = 545 [MH + A. Glucose-1P and galactose-1P (m / z = 259 [MH + B. Glucose-6P (m / z = 259 [MH + ]), and C. UDP-glucose (m / z = 565 [MH + ]). D. The normalized peak area of ​​UDP-glucose compared to the empty vector control (p-values ​​of two-sided t-test: Ptip: 0.01068, Pgap: 0.001356) and mass m / z = 545 [MH + ](p value of two-sided t-test: Ptip: 0.0412).

[0069] Fig.30 Relative transcript amounts of genes acbB, acbA, and acbV in the acbB overexpression mutant during the initial growth phase. Means and standard deviations of at least three biological replicates are shown. Differences from the empty vector control (set to value 1) were tested by two-sided t-tests (p values ​​from left to right correspond to pSETT4gap::acbB, pSETT4tip::acbB, pSETT4::P acbB :acbB, pSET152::P acbB :acbB): B :4.332e-05,4.561e-06,0.3511,0.7082; Aa :0.3384,0.0001164,0.5967,0.4246; acV :0.3033,0.0423,0.73,0.4687). Asterisks indicate significance levels: *p-value <α = 5%, **p-value <α = 1%, ***p-value <α = 0.1%.

[0070] Fig.31 Relative transcript amounts of the gene acbB in the acbB overexpressing mutants during the linear growth phase. Means and standard deviations of at least three biological replicates are shown. RT-qPCR indicated significant differences in gene expression compared to the empty vector control (value set to 1), which was tested by a two-sided t-test (p values ​​from left to right correspond to pSETT4gap::acbB, pSETT4tip::acbB, pSETT4::P acbB :acbB, pSET152::P acbB :acbB):acbB:0.02217,0.02771,0.03895,0.1582). Asterisks indicate significance levels: *p-value <α=5%, **p-value <α=1%, ***p-value <α=0.1%.

[0071] Fig.32 Growth and acarbose production of the gtaB overexpressing mutant in maltose-basic medium. The sampling times for RNA-isolation are indicated by arrows.

[0072] Fig.33 .Relative transcript amounts of gtaB in overexpression mutants. RT-qPCR showed a significant increase in gtaB expression compared to the empty vector control (value set to 1) (p-value of two-sided t-test: 0.01295). Asterisks indicate significance levels: *p-value <α=5%, **p-value <α=1.

[0073] Fig.34LC-MS analysis of intracellular metabolites of gtaB-overexpressing mutants. The mass m / z = 545 [MH + ](A), glucose-1P and galactose-1P (m / z=259[MH + ], B), glucose-6P (m / z = 259 [MH + ], C) and UDP-glucose (m / z = 565 [MH + ], D) peak area. For mass m / z = 545 [MH + ], a significant difference was observed compared with the empty vector control (p value of two-sided t-test: 0.01531). According to the two-sided t-test, all other peak areas were not significantly different.

[0074] A brief description of sequence ID

[0075] The sequence listing associated with the present application is submitted in electronic format and is incorporated by reference into this specification in its entirety.

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[0117] Detailed description

[0118] definition

[0119] Unless otherwise defined, all scientific and technical terms used in the specification, drawings and claims have the common meaning as commonly understood by those of ordinary skill in the art. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. If two or more documents incorporated by reference include disclosures that conflict and / or are inconsistent with each other, the document with a later effective date shall prevail. Materials, methods and embodiments are illustrative only and not restrictive. Unless otherwise stated, the following terms used herein (including specification and claims) have the definitions given below.

[0120] Unless the context clearly indicates otherwise, the terms "comprising," "including," "containing," "having," and the like are to be read expansively or open-endedly, without limitation. Unless otherwise indicated, singular forms such as "a," "an," or "the" include plural references. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, five, six, seven, eight, nine, ten or more elements.

[0121] It should also be understood that slight variations above and below the stated ranges can be used to achieve substantially the same results as the values ​​within the ranges. In addition, unless otherwise stated, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0122] In the case of providing a protein or amino acid sequence throughout the application, it will also be understood by those skilled in the art that single or multiple amino acids can be exchanged with amino acids having similar properties to achieve substantially the same effect, i.e., equivalent results. It will also be known to those skilled in the art that a determined protein or amino acid sequence can be encoded by a variety of different nucleic acid sequences. For a given amino acid sequence as defined herein, each of the countable nucleic acid sequences encoding a specific amino acid sequence should be considered as disclosed herein. In the case of providing a nucleic acid sequence throughout the application, it will also be understood that silent mutations can be introduced.

[0123] 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 cyclitol-containing aminoglycoside composed of a pseudodisaccharide and maltose α-1,4-glycosidically bound (Wehmeier and Piepersberg 2009). The pseudodisaccharide, named acarviose, is constructed from an unsaturated C7-aminocyclitol (also called valienol or valienamine) linked to C4 of 4,6-dideoxy-D-glucose via a nitrogen bond (see Figure 5 )(Wehmeier and Piepersberg 2009). This N-glycosidic bond cannot be hydrolyzed by exogenous α-1,4-glucoside hydrolases, resulting in an almost irreversible inhibition (Wehmeier and Piepersberg 2009; Brayer et al., 2000).

[0124] "Overexpression" of a gene product or protein as described herein refers to an increase in expression compared to a wild type or a specific reference strain. Preferably, a reference strain or control is a strain that is not engineered to specifically overexpress the corresponding gene or protein. For example, the control does not include a vector containing an expression cassette of the corresponding gene product or protein. For example, the overexpression of a gene product can be an increase during an early growth phase, a linear growth phase, a stationary phase, or an increase during any other time period. Preferably, overexpression is an increase of at least 1.5 times or at least 2 times compared to a control. With respect to transcript amounts, and if not otherwise defined herein, strong overexpression refers to log2 (fold change) > 6. With respect to transcript amounts, and if not otherwise defined herein, weak overexpression refers to log2 (fold change) < 2. With respect to transcript amounts, and if not otherwise defined herein, moderately strong overexpression refers to log2 (fold change) ≥ 2 and ≤ 6.

[0125] If the corresponding gene has been expressed by its gene product at all or expressed in a significantly reduced amount (e.g., less than 0.75 times or less than 0.5 times), the expression of the gene product or protein as described herein is "absent or reduced". If the gene product or protein has lost function, for example, in an instantaneous or permanent manner (e.g., by mutation or knocking down), the expression of the gene product or protein as described herein is also considered to be absent or reduced. Methods for monitoring the amount and / or activity of the gene product are known in the art and are also described in an exemplary manner herein. Generally, the appropriate method for obtaining the absence or reduction of gene product expression is a method for changing the genetic sequence or element of gene expression (e.g., by deletion or point mutation) and / or a method for negatively affecting the transcription and translation of the gene or the activity or half-life of the gene product (protein).

[0126] If not otherwise indicated, the symbol "Δ" refers to a "deletion mutant", ie a mutant in which a specific gene sequence is at least partially deleted.

[0127] The “early growth phase” is when the actinomycete strains are adapted to the culture medium and when the cell dry weight is below 3 g·L -1 time. After acclimatization, the culture metabolizes the nutrients supplied by the culture medium and begins to grow. Since Actinomycetes grow in a spherical mycelium that can only expand outside the sphere, the cells in the middle are isolated from nutrients and have only limited space for cell division. Therefore, only the cells in the outer layer of the spherical mycelium are dividing. Thus, the growth of Actinomycetes is linear rather than exponential - in contrast to other bacteria that grow as a single cell. The growth period is called the "linear growth period" for Actinomycetes and is measured at 3 g·L -1 The "stationary phase" is defined as the growth phase in which cells reach their capacity limits (for space and nutrients), respectively, and in which growth is reduced due to the formation of inhibitory byproducts or other chemical and physical factors (such as changes in osmotic pressure or pH). The stationary phase is the growth phase in which the number of dying cells equals the number of dividing cells. This phase is usually achieved at a cell dry weight of 16-18 g L in maltose basal medium. -1 Starts at .

[0128] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating nucleic acid molecules to which it is linked.

[0129] As used herein, the term "expression cassette" refers to a nucleic acid molecule comprising at least a gene and a regulatory sequence (eg, a promoter) for expression.

[0130] A "promoter" is a nucleic acid sequence that causes the initiation of transcription of a specific gene.

[0131] A "strong promoter" as defined herein is one that results in at least 5×10 -4 [L·g -1 ·min -1 ] and / or a promoter that results in 350-fold relative transcription (in log2 (fold change)) of the gusA gene compared to a promoter-free pGUS control vector. A detailed description of methods for characterizing promoter strength is provided in the Examples and (Schaffert et al. 2019).

[0132] Examples include the following promoters:

[0133] ·apm:9.2·10 -4 [L·g-1·min -1 ] and log2(fold change) = 360.78

[0134] ·ermE*:9.7·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 291.03

[0135] katE:5.1·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 342.51

[0136] ·moeE5:9.7·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 329.32

[0137] gapDH:11.5·10 -4 [L·g -1 ·min -1 ] and log2(fold change)=931.45, and

[0138] ·actP:22.9·10 -4 [L·g -1 ·min -1 ].

[0139] A "moderately strong promoter" as defined herein is one that results in at least 1·10 -4 [L·g -1 ·min -1], and / or a promoter that results in 10-fold relative transcription (in log2 (fold change)) of the gusA gene compared to a pGUS control vector without a promoter. Examples include the following promoters:

[0140] efp:3.1·10 -4 [L·g -1 min -1 ] and log2(fold change) = 53.08

[0141] ·cdaR:3.1·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 86.82

[0142] ·rpsL:3.5·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 98.53

[0143] ·rpsJ:3.7·10 -4 [L·g -1 ·min -1 ] and log2(fold change) = 123.97

[0144] ·cgt:2.5·10 -4 [L·g -1 ·min -1 ] and log2(fold change)=347.29, and

[0145] ·tipA:4.2·10 -4 [L·g -1 ·min -1 ] and log2(fold change)=191.

[0146] In some cases, moderately strong promoters resulted in at least 1·10 -4 [L·g -1 ·min -1 ] and maximum 5·10 -4 [L·g -1 ·min -1 ] of the normalized glucuronidase activity.

[0147] A weak promoter is defined as one that causes less than 1·10 -4 [L·g -1 ·min -1] and / or a promoter that results in less than 10-fold relative transcription compared to a promoterless pGUS control vector.

[0148] The term "Cgt" (ACSP50_5024, formerly: ACPL_5091) refers to an extracellular small carbohydrate binding protein, previously described as a cyclomaltodextrin glucanotransferase, due to the high similarity to the C-terminal domain of a cyclodextrin glycosyltransferase, obtained from actinomycetes such as 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 can be obtained via the UniProt identifier G8S155 (G8S155_ACTS5). Different isoforms and variants may exist in different strains, and are all included in the term. In the case where a specific mutation can be exchanged without changing the catalytic properties of the described initial sequence, it is apparent that the sequence with this functionally silent mutation is equivalent relative to the initial sequence. In addition, the protein may also be subjected to various modifications, such as synthetic or naturally occurring modifications.

[0149] The term "AcbB" (ACSP50_3608, formerly ACPL_3681) refers to a dTDP-D-glucose-4,6-dehydratase obtained from Actinomycetes (e.g., strain ATCC31044 / CBS 674.73 / SE50 / 110), which is likely involved in the biosynthesis of the acarviose portion of acarbose. The AcbB protein is encoded by the gene acbB. The sequence is described herein (SEQ ID No. 13) or can be obtained by the UniProt identifier Q9ZAE8 (RMLB_ACTS5). For different strains, different isoforms and variants may exist and are all included in the term. In cases where a particular mutation can be exchanged without changing the catalytic properties of the described initial sequence, it is clear that the sequence with such a functionally silent mutation is equivalent relative to the initial sequence. In addition, the protein can also undergo various modifications, such as synthetic or naturally occurring modifications.

[0150] The term "GtaB", also referred to as "GalU" (ACSP50_7820, formerly ACPL_7811), refers to a UTP-glucose-1-phosphate uridyltransferase obtained from Actinomycetes (e.g., strain ATCC 31044 / CBS 674.73 / SE50 / 110). GtaB appears to catalyze the interconversion of glucose-1P and UDP-glucose and may be involved in the precursor supply for acarbose. The GtaB protein is encoded by the gene gtaB. The sequence is described herein (SEQ ID No. 19) or can be obtained via the UniProt identifier G8S608 (ACPL_7811). For different strains, different isoforms and variants may exist and are all included in the term. In cases where a specific mutation can be exchanged without changing the catalytic properties of the described initial sequence, it is clear that the sequences with such functionally silent mutations are equivalent relative to the initial sequence. In addition, the protein may also be subjected to various modifications, such as synthetic or naturally occurring modifications.

[0151] As defined herein, "genes essential for carotenoid synthesis" are defined as genes absolutely required for the synthesis of carotenoids. Actinomycetes are known to produce a variety of soluble pigments, including yellow, orange and pink pigments of the carotenoid class. In actinomycetes, the gene set essential for carotenoid synthesis includes genes from the MEP / DOXP pathway, genes from terpene cluster 1, genes from terpene cluster 2a, genes from terpene cluster 2b, and genes from camphene-like monoterpene biosynthesis terpene cluster 3. The genes of the MEP / DOXP pathway include

[0152] i. 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID No. 23),

[0153] ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID No. 24),

[0154] iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID No. 25),

[0155] iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID No. 26),

[0156] v. 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID No. 27),

[0157] vi. 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase gene ispF (ACSP50_8046, SEQ ID No. 28), and / or

[0158] vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID No. 29).

[0159] The genes of terpene cluster 1 include

[0160] i. Isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID No.30),

[0161] ii. ζ-phytoene desaturase gene crtI (ACSP50_0147, SEQ ID No.10),

[0162] iii. polyisoprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID No.31),

[0163] iv. Phytoene synthase gene crtB (ACSP50_0149, SEQ ID No.32),

[0164] v. deoxyribonucleoside dipyrimidine photolyase gene (ACSP50_0150, SEQ ID No.33), or

[0165] vi. Pyridine nucleotide disulfide oxidoreductase gene (ACSP50_0151, SEQ ID No. 34).

[0166] The genes of terpene cluster 2a include

[0167] i. Transcription regulator gene (ACSP50_1631, SEQ ID No.35),

[0168] ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID No.36),

[0169] iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID No.37),

[0170] iv. polyisoprenyl synthase (farnesyl pyrophosphate synthase 2 gene) fps2 / crtE (ACSP50_1634, SEQ ID No.38), and

[0171] v. Methylenetetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID No. 39).

[0172] The genes of terpene cluster 2b include

[0173] i. LysR-family transcriptional regulator gene (ACSP50_1650, SEQ ID No.40),

[0174] ii. methyltransferase type 11 gene (ACSP50_1651, SEQ ID No.41),

[0175] iii. CDP-alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID No.42),

[0176] iv. ζ-phytoene desaturase (crtI-family) gene crtD (ACSP50_1653, SEQ ID No. 43),

[0177] v. glycosyltransferase gene cruC (ACSP50_1654, SEQ ID No.44),

[0178] vi. Hypothetical protein (membrane protein) gene cruF (ACSP50_1655, SEQ ID No.45),

[0179] vii. GCN5 family acetyltransferase gene (ACSP50_1656, SEQ ID No.46),

[0180] viii. monooxygenase gene (ACSP50_1657, SEQ ID No.47), and

[0181] ix. Short-chain dehydrogenase gene (ACSP50_1658, SEQ ID No.48).

[0182] Another gene necessary for carotenoid synthesis is the polyisoprene synthase gene crtE (ACSP50_3873, SEQ ID No. 49).

[0183] The genes of terpene cluster 3 for camphene-like monoterpene biosynthesis include

[0184] i. Transcription regulator (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID No.104),

[0185] ii. camphene synthase gene (ACSP50_1950, SEQ ID No.50),

[0186] iii. Methyltransferase (SAM-dependent) type 11 gene (ACSP50_1951, SEQ ID No.105),

[0187] iv. glycosyl hydrolase gene (ACSP50_1952, SEQ ID No.106), and

[0188] v. Oxidoreductase / aldehyde / ketoreductase (ACSP50_1953, SEQ ID No. 107).

[0189] Implementation

[0190] Although the Actinomycetales strain A. planarians SE 50 / 110 is used as a model strain for the present invention, it is clear to those skilled in the art that the general mechanisms and findings can be applied to other acarbose producing strains, such as those currently used for industrial production of acarbose. According to some embodiments, the Actinomycetales strain is a Micromonosporaceae strain. According to some embodiments, the Actinomycetales strain is an Actinomycetes strain. According to some embodiments, the Actinomycetales strain is an Actinomycetes strain. According to some embodiments, the Actinomycetales strain is Actinomycetes SE50 (ATCC 31042, CBS 961.70) (Frommer et al. 1973), Actinomycetes SE50 / 110 (ATCC31044, CBS 674.73) or an Actinomycetes strain derived therefrom. In some embodiments, the Actinomycetales strain is an Actinomycetes strain used commercially for acarbose production. In some embodiments, the Actinomycetales strain is an Actinomycetes strain commercially used for acarbose production, such as, for example, SN223-29-47, C445-P47, SN12755-38, SC3687-18-43, SC7177-40-17 or SN19910-37-21 disclosed in EP 2601209 B1 and CN103298828B, or a strain derived therefrom.

[0191] Improvement in acarbose production refers to an increase in the yield of acarbose in a specific time (total or relative to cell growth) and / or an improvement in the purity of acarbose, for example, a reduction in byproducts and / or acarbose analogs such as component C. The cultivation of the Actinomycetes strain can be performed as known in the art or as described herein. In some embodiments, the cultivation of the Actinomycetes strain is performed in a maltose-based medium.

[0192] According to a first aspect of the present invention, there is provided a method for engineering an Actinomycetes strain (eg, an Actinomyces strain) for improving acarbose production.

[0193] According to some first embodiments of the first aspect, the method according to the first aspect comprises engineering the Actinomycetales strain for the absence or reduced expression of the extracellular small carbohydrate binding protein Cgt (SEQ ID No. 20).

[0194] Surprisingly, the deletion of the carbohydrate binding protein Cgt (SEQ ID No. 20) resulted in improved production of acarbose. An increase in the final acarbose yield between 8.3% and 16.6% was achieved in three independent shake flask cultures (see Example "Δcgt shows improved acarbose formation on maltose-based medium", Fig.18 , Fig.19 , Table E10, Table E11).

[0195] Furthermore, the gene deletion mutant Δcgt performed better in screening experiments testing different carbon sources, or under carbon-limiting conditions (see Examples "Analysis of cgt expression during growth on different carbon sources", "Δcgt on different carbon sources or under carbon-limiting conditions", Fig.12 , Fig.13 , Fig.14 ), or under pH and osmotic stress (see Example "Δcgt has no effect on osmotic pressure tolerance or pH tolerance", Fig.15 , Fig.16 , Fig.17 ), showing no obvious growth phenotype. The inventors were also able to show that the absence of cgt had no negative effect on the expression of acarbose biosynthetic genes (see Example “Δcgt has no effect on the expression of acarbose biosynthetic genes”, Fig. 20 ).

[0196] Without being bound by theory, Cgt was found to be highly expressed in Actinoplanic SE50 / 110 based on a comprehensive study of the extracellular proteome (Wendler et al. 2013; Ortseifen 2016) and transcriptome (Schwientek et al. 2013). Its gene product is exported into the extracellular space, accounting for about 8% of the entire secreted proteome. The inventors analyzed the distribution of CBM-20 single-domain proteins in the prokaryotic kingdom by BlastP analysis. Interestingly, single CBM-20 domain proteins were found in only 17 other species (see Example "Distribution of single-domain CBM-20 proteins in the eubacterial kingdom"). Most of these were found in species of the Actinomycetales, such as in all strains of the genus Actinoplanic. Without being bound by theory, energy and resources (such as ATP and amino acids) are relieved by the absence or reduction of cgt expression. These resources can then be redirected to acarbose biosynthesis, which is a growth-related product.

[0197] According to some embodiments according to the first aspect, the method comprises the 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 establishment of CRISPR / Cas9 (Wolf et al. 2016) allow genome editing in Actinomycetes SE50 / 110. In some embodiments according to the first aspect, CRISPR / Cas9 technology can be used to engineer Actinomycetales strains so that expression is absent or reduced. In some embodiments, engineering Actinomycetales strains so that expression is absent or reduced can be performed as described in (Wolf et al. 2016). In some embodiments, Actinomycetales strains can be engineered so that expression is absent or reduced as described herein, for example, as described in the examples "Deletion of gene cgt by CRISPR / Cas9 technology" or "Deletion system based on homologous recombination and counter-selection with cytosine deaminase CodA".

[0198] For example, the present inventors have successfully established a new deletion system by homologous recombination, which uses an integrase-free vector backbone and CodA for counter-selection, as described in Zhao et al. (2017).

[0199] According to some second embodiments of the first aspect, the method according to the first aspect comprises engineering the Actinomycetales strain to eliminate or reduce the expression of at least one gene necessary for carotenoid synthesis. In some embodiments, the carotenoid is an orange pigment of Actinomycetes or a derivative thereof. In some different or identical embodiments, the carotenoid is a C40-carotenoid.

[0200] Engineering the Actinomycetales strain to have absent or reduced expression may be performed as previously described for the current aspect. According to some embodiments of the first aspect, the method comprises deletion or mutation of a gene necessary for carotenoid synthesis.

[0201] Actinomycetes are known to produce a variety of soluble pigments, including yellow, orange and pink pigments of the carotenoid class (Parenti and Coronelli 1979). The inventors observed that strong pigmentation was associated with a loss of acarbose production. This was confirmed by comparing the growth and acarbose yield of cultures exposed to light and protected from light (see Example "Light-dependent carotenoid formation and oxidative stress reduce acarbose production in Actinomycetes SE50 / 110", Fig. 22 ). While carotenoid formation was induced, acarbose production and growth of A. acarbose SE50 / 110 were strongly reduced when exposed to light ( Fig. 22). Overall, a 39% loss in the final acarbose concentration was monitored.

[0202] According to these findings, not only does it seem plausible that the pigment produced is not necessary (e.g. in a technical setting for commercial acarbose production), but also that reducing or depleting carotenoid synthesis in actinomycetes can be used to improve acarbose formation. To this end, the method according to the first aspect comprises reducing or depleting the expression of at least one gene necessary for carotenoid synthesis.

[0203] The inventors were also able to reconstruct carotenoid production in Actinoplanes SE50 / 110 (see Example "Functional correlation analysis of carotenoid formation", Figure 21). The gene set necessary for carotenoid synthesis in Actinoplanes includes genes from the MEP / DOXP pathway, genes from terpene cluster 1, genes from terpene cluster 2a, genes from terpene cluster 2b, and genes from terpene cluster 3 for camphene-like monoterpene biosynthesis.

[0204] According to some embodiments according to the current aspects and embodiments, at least one gene necessary for carotenoid synthesis is a gene of the MEP / DOXP pathway, e.g.

[0205] i. 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID No. 23),

[0206] ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID No. 24),

[0207] iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID No. 25),

[0208] iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID No. 26),

[0209] v. 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID No. 27),

[0210] vi. 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase gene ispF (ACSP50_8046, SEQ ID No. 28), and / or

[0211] vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID No. 29).

[0212] According to some embodiments according to the present aspects and embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 1, e.g.

[0213] i. isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID No.30),

[0214] ii. ζ-phytoene desaturase gene crtI (ACSP50_0147, SEQ ID No.10),

[0215] iii. polyisoprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID No.31),

[0216] iv. Phytoene synthase gene crtB (ACSP50_0149, SEQ ID No.32),

[0217] v. deoxyribonucleoside dipyrimidine photolyase gene (ACSP50_0150, SEQ ID No.33), or

[0218] vi. Pyridine nucleotide disulfide oxidoreductase gene (ACSP50_0151, SEQ ID No. 34).

[0219] According to some embodiments according to the current aspects and embodiments, at least one gene necessary for carotenoid synthesis is the ζ-phytoene desaturase gene crti (ACSP50_0147, SEQ ID No. 10). As mentioned above, carotenoid formation is not necessary under laboratory conditions. In order to improve acarbose production, interrupting the concurrent carotenoid biosynthesis pathway, in particular by deleting the central gene crtI, can be used for strain development.

[0220] According to some embodiments according to the present aspects and embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2a, e.g.

[0221] i. Transcription regulator gene (ACSP50_1631, SEQ ID No.35),

[0222] ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID No.36),

[0223] iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID No.37),

[0224] iv. polyisoprenyl synthase (farnesyl pyrophosphate synthase 2 gene) fps2 / crtE (ACSP50_1634, SEQ ID No.38), or

[0225] v. methylenetetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID No.39),

[0226] According to some embodiments according to the present aspects and embodiments, at least one gene essential for carotenoid synthesis is a gene of terpene cluster 2b, e.g.

[0227] i. LysR-family transcriptional regulator gene (ACSP50_1650, SEQ ID No.40),

[0228] ii. methyltransferase type 11 gene (ACSP50_1651, SEQ ID No.41),

[0229] iii. CDP-alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID No.42),

[0230] iv. ζ-phytoene desaturase (crtI-family) gene crtD (ACSP50_1653, SEQ ID No. 43),

[0231] v. glycosyltransferase gene cruC (ACSP50_1654, SEQ ID No.44),

[0232] vi. Hypothetical protein (membrane protein) gene cruF (ACSP50_1655, SEQ ID No.45),

[0233] vii. GCN5 family acetyltransferase gene (ACSP50_1656, SEQ ID No.46),

[0234] viii. monooxygenase gene (ACSP50_1657, SEQ ID No.47), or

[0235] ix. Short-chain dehydrogenase gene (ACSP50_1658, SEQ ID No.48),

[0236] According to some embodiments according to the current aspects and embodiments, the at least one gene necessary for carotenoid synthesis is the polyisoprenyl synthase gene crtE (ACSP50_3873, SEQ ID No. 49).

[0237] According to some embodiments according to the present aspects and embodiments, at least one gene essential for carotenoid synthesis is a gene of camphene-like monoterpene biosynthesis terpene cluster 3, e.g.

[0238] i. Transcription regulator (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID No.104),

[0239] ii. camphene synthase gene (ACSP50_1950, SEQ ID No.50),

[0240] iii. Methyltransferase (SAM-dependent) type 11 gene (ACSP50_1951, SEQ ID No.105),

[0241] iv. glycosyl hydrolase gene (ACSP50_1952, SEQ ID No.106), or

[0242] v. Oxidoreductase / aldehyde / ketoreductase (ACSP50_1953, SEQ ID No. 107).

[0243] Since carotenoids affect membrane fluidity, the lack of carotenoids, especially C40-carotenoids, may also affect the surface and hyphal structure of A. aeruginosa SE50 / 110. Regarding production, the disintegration of hyphal clumps is beneficial to increase the mycelial surface and the number of biochemically available cells.

[0244] According to some further embodiments, the method according to the first aspect comprises engineering an Actinomycetes strain for overexpression of the MerR- / HTH-transcriptional regulator gene merR (AcSP50_0145, SEQ ID No. 11). Engineering an Actinomycetes strain for overexpression may be performed as described elsewhere herein.

[0245] In addition to the genes mentioned as being essential for carotenoid synthesis, the inventors surprisingly identified a transcriptional repressor of carotenoid synthesis in 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, acarbose production was found to be reduced. When irradiated, both the wild type and ΔmerR were strongly colored, and the final acarbose concentrations of the two strains were similar, reaching about 0.52 g·L -1(Figure 24B and D). Under dark conditions, this corresponds to a 38% reduction in acarbose formation compared to the wild type (to 0.83 g·L -1 ). This is consistent with previous wild-type growth experiments.

[0246] Under dark conditions, ΔmerR produced about 15% less acarbose (0.70 g·L -1 Without being bound by theory, these production losses are believed to be caused by a waste of resources caused by carotenoid formation in the deletion mutants (Figure 24C). In summary, the yield loss (38-39%) under light conditions may be attributed to further light-induced stress in the deletion mutants and the wild type.

[0247] According to some third embodiments according to the first aspect, the method comprises engineering an Actinomycetales strain to overexpress dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13).

[0248] According to the present invention, it was surprisingly found that overexpression of the acb gene encoding the dTDP-D-glucose-4,6-dehydratase AcbB significantly increased the final acarbose concentration by about 50%. This is particularly surprising because other genes of the Acb cluster, such as AcbC, do not lead to improved acarbose formation. Moreover, the observed increase is superior to the increase observed by overexpression of the complete acb cluster as described by Zhao et al. (Zhao, Xie, et al. 2017).

[0249] According to some embodiments, the strains do not include engineered Actinomycetales strains for overexpression of other genes in the acb cluster besides AcbA.

[0250] dTDP-D-glucose-4,6-dehydratase AcbB appears to be involved in the production of activated amino sugars from D-glucose-1P, which is the feeder pathway for acarbose biosynthesis ( Figure 1 ): Without wishing to be bound by theory, it was surprisingly found that increased AcbB activity also improved the supply of modified precursors.

[0251] As described herein, overexpression of AcbB refers to an increase in AcbB expression compared to the wild type or a specified reference strain / control. For example, overexpression of a gene product can be increased in the early growth phase, the linear growth phase, the stationary phase, or during any other time period.

[0252] Preferably, as described herein, overexpression of AcbB refers to an increase of at least 1.5-fold or at least 2-fold in AcbB transcript and / or protein compared to a control. With respect to the amount of AcbB transcripts, if not otherwise defined herein, strong overexpression refers to log2 (fold change) > 6. With respect to the amount of AcbB transcripts, if not otherwise defined herein, moderately strong overexpression refers to log2 (fold change) ≥ 2 and ≤ 6.

[0253] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by at least 1.5 or at least 2 log2 (fold change) during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0254] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) ≥ 2 and ≤ 6 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period such as during the early growth phase and / or during the linear growth phase.

[0255] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0256] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) > 6 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0257] In overexpression mutants with expression vectors containing heterologous promoters, the relative transcription of acbB between the two sampling times decreased from 4.06-fold to 3.33-fold (log2(fold change)) in pSETT4tip::acbB (medium strength promoter) and from 6.54-fold to 2.05-fold in pSETT4gap::acbB (strong promoter) (see Example "Moderate overexpression of acbB leads to improved acarbose formation").

[0258] According to some embodiments, engineering an Actinomycetales strain for gene overexpression according to the first aspect can be performed by any method known in the art or described herein.

[0259] As described in the example "Moderate overexpression of AcbB leads to improved acarbose formation", two PSET4-based overexpression mutants were generated, in which acbB was transcribed under the control of the moderately strong TipA promoter or the strong gapDH promoter. The native promoter was used in the pSET152-vector and pSET4-vector backgrounds as controls. In particular, the mutants with acbB transcribed under the control of the heterologous tipA promoter showed enhanced acarbose production compared to the control strain ( Fig. 27 , Fig.28 Compared to the empty vector control, the yield coefficients increased to 48.6% and 51.9%. By using the strong gapDH-promoter, it was found that the yield coefficient of acarbose was slightly increased ( Fig.28 ).

[0260] According to some embodiments, engineering an Actinomycetales strain for overexpression of a gene according to the first aspect can be performed by introducing a vector comprising an expression cassette for AcbB (SEQ ID No. 13) into the Actinomycetales strain. In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. If a vector comprises at least one, two, three, or four elements of another vector, the vector is derived from the second vector.

[0261] According to some embodiments, engineering an Actinomycetes strain for overexpressing a gene according to the first aspect can be performed by introducing a vector comprising an expression cassette for AcbB (SEQ ID No. 13) into the Actinomycetes strain. In some of these or other embodiments, the expression cassette is under the control of a moderately strong promoter, wherein the moderately strong promoter is characterized by a normalized glucuronidase activity of at least 1×10 -4 , preferably in the range of 1×10 -4 With 5×10 -4 [Lg -1 min -1], e.g., as described elsewhere herein. In some embodiments, the promoter is selected from the group consisting of efp promoter (SEQ ID No.92), cdaR promoter (SEQ ID No.97), rpsL promoter (SEQ ID No.99), rpsJ promoter (SEQ ID No.93), cgt promoter (SEQ ID No.91), or tipA promoter (SEQ ID No.81). In some embodiments, the promoter is tipA promoter (SEQ ID No.81). Excellent results for acarbose production were obtained with pSETT4tip::acbB, see Fig. 27 , Fig.28 .

[0262] In some embodiments, the expression cassette is under the control of a strong promoter, such as one characterized by a normalized glucuronidase activity of at least 5×10 -5 [L g -1 min -1 ], for example as described elsewhere herein. In some embodiments, the promoter is selected from apm promoter (SEQ ID No.96), ermE* promoter (SEQ ID No.98), katE promoter (SEQ ID No.94), moeE5 promoter (SEQ ID No.95) or gapDH promoter (SEQ ID No.82).

[0263] According to some embodiments, the method according to the first aspect comprises engineering an Actinomycetales 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, if not explicitly stated otherwise, which are also compatible with all other embodiments described herein, for Acb genes other than AcbB and AcbA, genetic engineering does not result in an increase in transcripts and / or proteins of log2 (fold change) ≥ 2. In some embodiments that are also compatible with all other embodiments described herein, genetic engineering does not result in an increase in transcripts and / or proteins of AcbC of log2 (fold change) ≥ 2.

[0264] Upon overexpression of AcbB, other genes of the acb gene cluster were not significantly affected, for example in the early growth phase, as shown for acbA and acbV ( Fig.30 The only exception was that the transcript abundance of acbA was slightly higher in pSETT4tip::acbB (log2(fold change) = 1.87).

[0265] According to some embodiments, the method according to the first aspect comprises engineering an Actinomycetales strain for overexpressing AcbB (SEQ ID No. 13) and AcbS (ACSP50_3596) and / or Acbl (ACSP50_3599).

[0266] The transfer reaction of amino sugars to cyclitol precursors can be enhanced by (additional) overexpression of AcbS and / or AcbI. Figure 1 ), the reaction is catalyzed by AcbS (ACSP50_3596) or AcbI (ACSP50_3599).

[0267] According to some embodiments, the method according to the first aspect comprises engineering an Actinomycetales strain for overexpressing 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 appears to enhance the synthesis of both acarbose chains.

[0268] According to some fourth embodiments of the first aspect, the method comprises engineering an Actinomycetales strain to overexpress UDP-glucose-1P uridyltransferase GtaB (SEQ ID No. 19).

[0269] By moderate overexpression of gtaB, an increase of 8.5% in the final acarbose concentration was observed, see Example "Moderate overexpression of gtaB leads to improvement in acarbose formation", Fig.32 , Fig.33 Interestingly, acarbose formation increased specifically during the late linear growth phase to the stationary growth phase ( Fig.32 Without being bound by theory, this may be due to improved distribution of the precursor glucose-1P (see Fig.34 ).

[0270] Overexpression of GtaB (SEQ ID No. 19) as described herein refers to an increase in the expression of a GtaB transcript and / or protein compared to the wild type or a specified reference strain / control. For example, overexpression of a gene product can be an increase during the early growth phase and / or during the linear growth phase and / or during the stationary phase, and / or during any other time period.

[0271] Preferably, overexpression is an increase of at least 1.5-fold or at least 2-fold in GtaB transcript and / or protein compared to a control. With respect to the amount of GtaB transcripts, if not otherwise defined herein, strong overexpression means log2 (fold change) > 6. With respect to the amount of GtaB transcripts, if not otherwise defined herein, moderately strong overexpression means log2 (fold change) ≥ 2 and ≤ 6.

[0272] According to some embodiments, overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in expression of GtaB by at least 1.5 or at least 2 log2 (fold change) during the early growth phase and / or during the linear growth phase and / or during the stationary phase, and / or an increase during any other time period.

[0273] In one of the overexpression mutants described herein, the relative transcript amount of the gene gtaB increased 2.64-fold (log2(fold change)) ( Fig.33 ).

[0274] According to some embodiments, the overexpression of UDP-glucose-1P uridyl transferase GtaB is an increase in the expression of GtaB transcripts and / or proteins by log2 (fold change) ≥2 and ≤6 during the early growth phase and / or during the linear growth phase and / or during the stationary phase, and / or an increase during any other time period. According to some embodiments, the overexpression of UDP-glucose-1P uridyl transferase GtaB is an increase in the expression of GtaB by log2 (fold change) ≥3 and ≤5 during the early growth phase and / or during the linear growth phase and / or during the stationary phase, and / or an increase during any other time period.

[0275] According to some embodiments, the overexpression of UDP-glucose-1P uridyltransferase GtaB is an increase in the expression of GtaB by log2 (fold change) ≥ 6 during the early growth phase and / or during the linear growth phase and / or during the stationary phase.

[0276] According to some embodiments, the engineered Actinomycetes strain according to the first aspect for gene overexpression can be carried out by introducing a vector containing an expression cassette of GtaB (SEQ ID No. 19) into the Actinomycetes strain. In some embodiments, the expression vector is derived from pSET152. In some embodiments, the expression vector is derived from pSETT4. If a vector comprises at least one, two, three, or four elements of another vector, the vector is derived from the second vector.

[0277] According to some embodiments, engineering the Actinomycetes strain for gene overexpression according to the first aspect may be performed by introducing a vector comprising an expression cassette of GtaB (SEQ ID No. 19) into the Actinomycetes strain.

[0278] In some of these or other embodiments, the expression cassette is under the control of a moderately strong promoter characterized by a normalized glucuronidase activity of at least 1×10 -4 Up to 5×10 -5 [Lg -1 min -1 ], e.g., as described elsewhere herein. In some embodiments, the promoter is selected from the group consisting of efp promoter (SEQ ID No.92), cdaR promoter (SEQ ID No.97), rpsL promoter (SEQ ID No.99), rpsJ promoter (SEQ ID No.93), cgt promoter (SEQ ID No.91), or tipA promoter (SEQ ID No.81). In some embodiments, the promoter is tipA promoter (SEQ ID No.81). For example, good results for acarbose production were obtained with pSETT4tip::gtaB, see Fig.32 , Fig.33 .

[0279] In some embodiments, the expression cassette is under the control of a strong promoter, such as one characterized by a normalized glucuronidase activity of at least 5×10 -5 [L·g -1 ·min -1 ], for example as described elsewhere herein. In some embodiments, the promoter is selected from apm promoter (SEQ ID No.96), ermE* promoter (SEQ ID No.98), katE promoter (SEQ ID No.94), moeE5 promoter (SEQ ID No.95) or gapDH promoter (SEQ ID No.82).

[0280] According to some further or identical embodiments of the first aspect, the method comprises engineering an Actinomycetales strain for moderate overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) and GtaB (SEQ ID No. 19).

[0281] Surprisingly, it was found that overexpression of GtaB triggers an improvement in acarbose formation. By moderate overexpression of acbB (e.g. by using the tipA-promoter), a positive effect on acarbose production was observed, with about 50% more acarbose being obtained in two independent cultivations. Thus, an improvement in acarbose biosynthesis was obtained by overexpression of a single acb gene, AcbB. Furthermore, by moderate overexpression of gtaB, an increase of 8.5% in the final acarbose concentration was observed. It seems reasonable that by combined overexpression of acbB and gtaB, the flux through amino sugar biosynthesis is improved, leading to a further enhancement of acarbose production.

[0282] Without being bound by theory, strong overexpression of AcbB induces only a smaller increase in acarbose production compared to moderately strong overexpression of AcbB. This may be due to the imbalance in glucose-phosphate metabolism that occurs when AcbB is overexpressed in large amounts. Overexpression of gtaB can eliminate this imbalance, so it seems reasonable that combined overexpression of both acbB and gtaB causes a further increase in acarbose production.

[0283] Interestingly, the mass m / z = 545 [MH + ] was significantly reduced (by about 48%), which may correspond to dTDP-4-keto-6-deoxy-D-glucose, the proposed product of AcbB. This may indicate that the flow through the synthetic chain is more balanced, because the accumulation of this metabolite is reduced compared with the empty vector control and the AcbB overexpression mutant ( Fig.34 ).

[0284] According to some embodiments, the method according to the first aspect comprises engineering an Actinomyces strain to

[0285] (i) for eliminating or reducing the expression of extracellular small carbohydrate binding protein Cgt (SEQ ID NO. 20), and / or,

[0286] (ii) for the absence or reduction of expression of at least one gene involved in carotenoid synthesis, and / or,

[0287] (iii) for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13), and / or

[0288] (iv) for overexpression of UDP-glucose-1P uridyltransferase GtaB (SEQ ID No. 19).

[0289] According to some embodiments, the method according to the first aspect further comprises engineering the Actinomycetales strain for the absence or reduced expression of treY.

[0290] According to some embodiments, the method according to the first aspect further comprises

[0291] (i) deletion or mutation of the gene encoding the extracellular small carbohydrate binding protein Cgt (SEQ ID No. 20), and / or

[0292] (ii) deletion or mutation of at least one gene involved in carotenoid biosynthesis, and / or

[0293] (iii) introducing a vector containing an expression cassette of AcbB (SEQ ID No. 13) into an Actinomycetales strain, and / or

[0294] (iv) introducing a vector containing the expression cassette of GtaB (SEQ ID No. 19) into an Actinomycetales strain.

[0295] According to some embodiments, the expression cassette according to (iii) and / or (iv) is under the control of a moderately strong promoter, characterized in that in a glucuronidase assay, the normalized glucuronidase activity is between 1×10 -4 and 5×10 -5 [L·g -1 ·min -1 ]between.

[0296] According to a second aspect, an Actinomycetes strain, such as an Actinomyces strain, for producing acarbose is provided. According to some embodiments, the Actinomycetes strain is a strain produced by a method according to the first aspect. According to some other embodiments, the Actinomycetes strain is genetically engineered so that the expression of the extracellular small carbohydrate binding protein Cgt (SEQ ID NO.20) is absent or reduced. According to some embodiments, the Actinomycetes strain is a Δcgt mutant. The Δcgt mutant is an Actinomycetes strain variant in which the gene Cgt (SEQ ID No.20) has been at least partially deleted or inverted.

[0297] According to some of these or other embodiments, the Actinomycetales strain is genetically engineered to have absent or reduced expression of at least one gene necessary for carotenoid synthesis. According to some embodiments, at least one gene necessary for carotenoid synthesis has been at least partially deleted or inverted. According to some of these or other embodiments, at least one gene necessary for carotenoid synthesis comprises at least one gene selected from any of the following:

[0298] a. Genes of the MEP / DOXP pathway, e.g.

[0299] i. 1-deoxy-D-xylulose-5-phosphate synthase gene dxs (ACSP50_7096, SEQ ID No. 23),

[0300] ii. 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gene ispG (ACSP50_7248, SEQ ID No. 24),

[0301] iii. 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (ACSP50_7250, SEQ ID No. 25),

[0302] iv. 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene ispH (ACSP50_7707, SEQ ID No. 26),

[0303] v. 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase gene ispE (ACSP50_7802, SEQ ID No. 27),

[0304] vi. 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase gene ispF (ACSP50_8046, SEQ ID No. 28), and / or

[0305] vii. 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene ispD (ACSP50_8047, SEQ ID No. 29),

[0306] b. Genes of terpene cluster 1, e.g.

[0307] i. Isopentenyl-diphosphate delta-isomerase gene idi (ACSP50_0146, SEQ ID No.30),

[0308] ii. ζ-phytoene desaturase gene crtI (ACSP50_0147, SEQ ID No.10),

[0309] iii. polyisoprenyl synthase gene crtE / ldsA (ACSP50_0148, SEQ ID No.31),

[0310] iv. Phytoene synthase gene crtB (ACSP50_0149, SEQ ID No.32),

[0311] v. deoxyribonucleoside dipyrimidine photolyase gene (ACSP50_0150, SEQ ID No.33), or

[0312] vi. Pyridine nucleotide disulfide oxidoreductase gene (ACSP50_0151, SEQ ID No.34)

[0313] c. Genes of terpene cluster 2a, e.g.

[0314] i. Transcription regulator gene (ACSP50_1631, SEQ ID No.35),

[0315] ii. Lycopene cyclase gene (ACSP50_1632, SEQ ID No.36),

[0316] iii. Lycopene cyclase gene (ACSP50_1633, SEQ ID No.37),

[0317] iv. polyisoprenyl synthase (farnesyl pyrophosphate synthase 2 gene) fps2 / crtE (ACSP50_1634, SEQ ID No.38), or

[0318] v. methylenetetrahydrofolate reductase (NADPH) gene (ACSP50_1635, SEQ ID No.39),

[0319] d. Genes of terpene cluster 2b, e.g.

[0320] i. LysR-family transcriptional regulator gene (ACSP50_1650, SEQ ID No.40),

[0321] ii. methyltransferase type 11 gene (ACSP50_1651, SEQ ID No.41),

[0322] iii. CDP-alcohol phosphatidyltransferase pgsA (ACSP50_1652, SEQ ID No.42),

[0323] iv. ζ-phytoene desaturase (crtI-family) gene crtD (ACSP50_1653, SEQ ID No. 43),

[0324] v. glycosyltransferase gene cruC (ACSP50_1654, SEQ ID No.44),

[0325] vi. Hypothetical protein (membrane protein) gene cruF (ACSP50_1655, SEQ ID No.45),

[0326] vii. GCN5 family acetyltransferase gene (ACSP50_1656, SEQ ID No.46),

[0327] viii. Monooxygenase gene (ACSP50_1657, SEQ ID No.47),

[0328] ix. Short-chain dehydrogenase gene (ACSP50_1658, SEQ ID No.48),

[0329] e. polyisoprene synthase gene crtE (ACSP50_3873, SEQ ID No.49), or

[0330] f. Genes of camphene-like monoterpene biosynthesis terpene cluster 3, e.g.

[0331] i. Transcription regulator (Crp / Fnr family) gene eshA (ACSP50_1949, SEQ ID No.104),

[0332] ii. camphene synthase gene (ACSP50_1950, SEQ ID No.50),

[0333] iii. Methyltransferase (SAM-dependent) type 11 gene (ACSP50_1951, SEQ ID No.105),

[0334] iv. glycosyl hydrolase gene (ACSP50_1952, SEQ ID No.106),

[0335] v. Oxidoreductase / aldehyde / ketoreductase (ACSP50_1953, SEQ ID No. 107).

[0336] According to some of these or other embodiments, the Actinomycetales strain is genetically engineered to overexpress the MerR- / HTH-transcriptional regulator gene merR (ACSP50_0145, SEQ ID No. 11).

[0337] According to some of these or other embodiments, the Actinomycetales strain is genetically engineered to overexpress dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13).

[0338] As described elsewhere herein, overexpression of AcbB refers to an increase of AcbB by at least 1.5-fold or at least 2-fold compared to a control. Preferably, the control is a strain that has not been engineered to specifically overexpress 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.

[0339] For example, overexpression of a gene product can be an increase during an early growth phase, during a linear growth phase, during a stationary phase, or during any other time period.

[0340] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by at least 1.5 or at least 2 log2 (fold change) during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0341] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID NO. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) ≥ 2 and ≤ 6 during the early growth phase, during the linear growth phase, during the stationary phase, or at any other time such as during the early growth phase and / or the linear growth phase.

[0342] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0343] According to some embodiments, overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) is an increase in expression of AcbB transcript and / or protein by log2 (fold change) > 6 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0344] According to some embodiments, the strain of Actinomycetales genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises a vector for overexpression of AcbB. According to some of these embodiments, the vector is as described herein, preferably according to one aspect described herein.

[0345] According to some embodiments, the Actinomycetales strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises an expression cassette of AcbB (SEQ ID No. 13) under the control of a moderately strong promoter.

[0346] According to some embodiments, the Actinomycetales strain genetically engineered for overexpression of dTDP-D-glucose-4,6-dehydratase AcbB (SEQ ID No. 13) comprises an expression cassette of AcbB (SEQ ID No. 13) under the control of a strong promoter. Preferably, the promoter is not the native promoter of AcbB.

[0347] According to some of these or other embodiments, the Actinomycetales strain is genetically engineered to overexpress UDP-glucose-1P uridyltransferase GtaB (SEQ ID No. 19).

[0348] Overexpression of GtaB (SEQ ID No. 19) as described elsewhere herein refers to an increase in the expression of GtaB compared to the wild type or a specified reference strain / control. Preferably, the control is a strain that has not been engineered to specifically overexpress GtaB (SEQ ID No. 19). For example, the control does not contain a vector containing an expression cassette of GtaB (SEQ ID No. 19). For example, overexpression of the gene product can be an increase during the early growth phase and / or during the linear growth phase and / or during the stationary phase, and / or during any other time period.

[0349] According to some embodiments, overexpression of GtaB is an increase in expression of GtaB transcript and / or protein by at least 1.5 or at least 2 log2 (fold change) during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0350] According to some embodiments, overexpression of GtaB is an increase in expression of GtaB transcript and / or protein by log2 (fold change) ≥ 2 and ≤ 6 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period, such as during the early growth phase and / or during the linear growth phase.

[0351] According to some embodiments, overexpression of GtaB is an increase in expression of GtaB transcript and / or protein by log2 (fold change) >3 and <5 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0352] According to some embodiments, overexpression of GtaB is an increase in expression of GtaB transcript and / or protein by log2 (fold change) > 6 during the early growth phase, during the linear growth phase, during the stationary phase, or during any other time period.

[0353] According to some embodiments, the Actinomycetales strain genetically engineered for overexpression of GtaB comprises a vector for overexpression of GtaB. According to some of these embodiments, the vector is as described herein, preferably according to one aspect described herein.

[0354] According to some embodiments, the Actinomycetales strain genetically engineered to overexpress GtaB (SEQ ID No. 19) comprises an expression cassette of GtaB (SEQ ID No. 19) under the control of a moderately strong promoter.

[0355] According to some embodiments, the Actinomycetales strain genetically engineered for overexpression of GtaB (SEQ ID No. 19) comprises an expression cassette of GtaB (SEQ ID No. 19) under the control of a strong promoter. Preferably, the promoter is not the native promoter of GtaB.

[0356] According to a third aspect, there is provided an Actinomyces strain for producing acarbose, such as an Actinomyces strain, for producing acarbose.

[0357] According to some embodiments, there is provided a method for producing acarbose, wherein the method comprises using the Actinomyces strain according to the second aspect.

[0358] For genetic engineering of actinomycetes, expression systems are needed to overexpress single or multiple genes.According to a fourth aspect, an expression vector for actinomycetes is provided.

[0359] According to some embodiments, the vector according to the fourth aspect comprises a moderately strong promoter, characterized in that the normalized glucuronidase activity in a glucuronidase assay is at least 1×10 -4 [L·g -1 ·min -1 In some embodiments, the medium strong promoter is selected from efp according to SEQ ID No.92, cdaR according to SEQ ID No.97, rpsL according to SEQ ID No.99, rpsJ according to SEQ ID No.93, cgt according to SEQ ID No.91 or tipA according to SEQ ID No.81.

[0360] According to some embodiments, the vector according to the fourth aspect comprises a strong promoter, characterized in that the normalized glucuronidase activity in a glucuronidase assay is at least 5×10 -4 [L·g -1 ·min -1In some embodiments, the strong promoter is selected from apm according to SEQ ID No.96, ermE* according to SEQ ID No.98, katE according to SEQ ID No.94, moeE5 according to SEQ ID No.95 or gapDH according to SEQ ID No.82.

[0361] In order to find other suitable promoters that allow moderate to strong gene expression, promoter screening can be performed using 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.

[0362] 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.

[0363] In some embodiments, the expression cassette may also contain the lacZα-gene under the control of the lac-promoter. The lacZα-gene encodes the catalytic domain of β-galactosidase, which enables rapid selection of the integration of the target sequence by blue / white selection in the cloning strain E. coli DH5αMCR (NC_017638.1) (Grant et al. 1990).

[0364] Without being bound by theory, the vector according to the current aspect comprises elements for vector replication, transfer, maintenance and selection. In some embodiments, at least one of these elements is derived from pSET152.

[0365] In some embodiments, the vector according to the current aspect comprises a sequence portion of the pSET152 vector of Bierman et al. (1992).

[0366] Preferably, the vector does not contain putative antisense promoters according to SEQ ID NO 108 and / or SEQ ID NO 109. These antisense promoters were identified by the inventors by sequencing of 5'-primary transcript libraries and compromise the suitability of the vector pSET 152. Briefly, identification was performed by sequencing of enriched primary transcript libraries. Two putative promoters were identified behind the gene of interest in antisense orientation ( Fig.26 ). The two pseudopromoters were removed to prevent antisense transcription.

[0367] In addition, a T4 terminator was introduced after the expression cassette in the reverse orientation to prevent further putative antisense reading (see e.g. Figure 6 ). In some embodiments, the vector comprises at least one T4 terminator (derived from bacteriophage T4). The T4-terminator can effectively block transcription and prevent read-through from the integrase gene into the gene of interest. In some embodiments, the vector comprises a T4 terminator in the opposite direction behind the expression cassette to prevent further putative antisense reading. 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 the expression cassette and two after the expression cassette.

[0368] In some embodiments, the vector comprises Integrase gene int. In some of these embodiments, The integrase gene int is derived from pSET152. In some embodiments, the vector according to the first aspect further comprises a ligation site attP. The integrase of the integrase gene int mediates the integration of the vector into the host chromosome at different genomic locations by catalyzing the targeted and unidirectional recombination of two junction sites: attP located on the vector and attB located in the gene ACSP50_6589 in the host chromosome (former: ACPL_6602) (Te Poele et al. 2008; Gren et al. 2016). Without being bound by theory, after integration, the vector is flanked by a left junction site (attL) and a right junction site (attR), which originates from attP-attB-recombination (te Poele, Bolhuis und Dijkhuizen 2008).

[0369] In some embodiments, the vector comprises a transfer origin such as a transfer origin (incP) and / or a relaxosome gene such as the relaxosome gene traJ. In some of these embodiments, the transfer origin such as the transfer origin (IncP) and / or the relaxosome gene such as traJ is derived from pSET152. The transfer origin and relaxosome gene enable the plasmid to be transferred from a donor strain (e.g., E. coli ET12567 / pUZ8002 (Kieser et al. 2000)).

[0370] 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 the plasmid to replicate in a cloning strain (E. coli DH5αMCR) and a donor strain (E. coli ET12567 / pUZ8002).

[0371] In some embodiments, the vector according to the first aspect comprises at least one resistance marker, such as a resistance marker mediating apramycin resistance (aac(3)IV, apmR). The resistance marker mediating apramycin resistance (aac(3)IV, apmR) can be used for selection.

[0372] According to some embodiments of the fourth aspect, the expression vector comprises at least one element of pSET152, such as (a) according to SEQ ID No. 85 integrase gene int, (b) transfer origin (incP) according to SEQ ID No.87, (c) relaxosome gene traJ according to SEQ ID No.88, or (d) high copy number ColE1 / pMB1 / pBR322 / pUC according to SEQ ID No.89, and further does not contain putative antisense promoters according to SEQ ID No.108 and SEQ ID No.109.

[0373] According to some embodiments of the fourth aspect, the expression vector comprises (a) the ΦC31 integrase gene int according to SEQ ID No.85, and (b) a transfer origin (incP) according to SEQ ID No.87, and (c) a relaxosome gene traJ according to SEQ ID No.88, and (d) an origin of replication, for example the high copy number ColE1 / pMB1 / pBR322 / pUC, origin of replication (ori) according to SEQ ID No.89, and (e) optionally at least one resistance marker, for example a resistance marker mediating apramycin resistance, for example aac(3)IV, apmR according to SEQ ID No.90, and (f) optionally at least one T4-terminator, and (g) optionally, wherein the vector does not comprise a putative antisense promoter according to SEQ ID No.108 and / or SEQ ID No.109.

[0374] According to some embodiments, the vector comprises a sequence according to SEQ ID No. 110 or SEQ ID No. 111. According to some embodiments, the vector comprises a sequence according to SEQ ID No. 110 or SEQ ID No. 111 or a fragment thereof.

[0375] In some embodiments, the advantage of the vector is a simple cloning mechanism that allows the integration of different promoters. Thus, the system can be quickly adapted to other species, such as acarbose production strains. Example

[0376] General tools and methods

[0377] Strains and plasmids

[0378] All strains used in this study are listed in Table E1. Recombinant strains used and generated in this study are listed in Table E2, Table E3 and Table E4 (plasmid-based expression systems are listed in Table E2, deletion and integration constructs cloned and stored in E. coli DH5αMCR are listed in Table E3, and deletion and integration mutants of Actinomycetes SE50 / 110 are listed in Table E4).

[0379] Table E1 Culture collection of microorganisms

[0380]

[0381]

[0382] Table E2 Replication and integration vector systems

[0383]

[0384]

[0385] Table E3 Vector system for targeted deletion and integration based on pCRISPomyces-2 of Cobb et al. (2015)

[0386]

[0387] Table E4 Deletion and integration mutants obtained by CRISPR / Cas9 technology in Actinomycetes ssp.

[0388]

[0389]

[0390] Culture medium and culture conditions

[0391] Unless otherwise stated, all chemicals and media components were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany), Sigma-Aldrich (St. Louis, USA), SERVA Electrophoresis GmbH (Heidelberg, Germany), or VWR International (Pennsylvania, USA).

[0392] Preparation of glycerol stock of Actinomycetes SE50 / 110

[0393] To prepare glycerol stocks, Actinomycetes SE50 / 110 (ATCC 31044) was cultured in complex medium NBS (11 g L -1 Glucose 1H2O, 4g·L -1 Peptone, 4 g L -1 Yeast extract, 1g·L -1 MgSO4·7H2O, 2g·L - 1 KH2PO4, 4g·L -1 Glycerol stocks were stored at -80 °C.

[0394] Growth on solid medium and preparation of spore solution

[0395] For sporulation, 200-300 μL of the glycerol stock was plated on soy flour medium (SFM-agar) (20 g L -1 Soybean flour( Naturkost (Cologne, Germany)), 20g·L -1 D-mannitol, 20 g·L -1 Bacto TM Agar (Becton-Dickinson, Heidelberg, Germany), 167 μL of 10 N NaOH in tap water) was grown on agar plates. After 5-7 days of incubation at 28°C, spores can be harvested by washing with a cotton swab in 3 ml of ddH2O as described by Wolf et al. (2016).

[0396] Preparation of basal medium

[0397] Preparation of maltose basal medium (72.06 g·L -1 Maltose·1H2O,5g·L -1 (NH4)2SO4, 0.184 g·L-1 FeCl2·4H2O,5.7g·L -1 Na3C6H5O7·2H2O,1g·L -1 MgCl2·6H2O,2g·L -1 CaCl2·2H2O, trace elements (final concentrations: 1 μM CuCl2, 50 μM ZnCl2, 7.5 μM MnCl2, dissolved in 1 M HCl) and 5 g·L -1 Phosphate buffer consisting of 100% K2HPO4 and 100% KH2PO4 was used and sterilized by filtration according to the protocol of Wendler et al. (2013).

[0398] In order to replace the carbon source maltose, 79.2 g·L -1 Glucose·1H2O, 72.0g·L -1 C-pur (Cerestar 01908, Cerestar GmbH, Krefeld, Germany), 71.9g·L -1 Galactose, 68.4 g·L -1 Cellobiose, 71.9 g·L -1 D-arabinose or 72.0 g·L -1 D-lactose replaced maltose monohydrate.Mixtures of maltose and glucose were prepared in ratios of 90:10, 80:20 and 50:50 (v / v).

[0399] For the starch medium, a 4% (w / v) milky solution of "soluble starch" from Acros Organics (Division of Thermo Fisher Scientific, Geel, Belgium) was produced. To this end, sterile water was preheated to 90°C in a water bath and the weighed starch portion was added under stirring. Then, the remaining medium components were added. In order to allow comparison with the starch culture, a 4% (w / v) milky solution of "soluble starch" from Acros Organics (Division of Thermo Fisher Scientific, Geel, Belgium) was produced. To this end, sterile water was preheated to 90°C in a water bath and the weighed starch portion was added under stirring. Then, the remaining medium components were added. To allow comparison with the starch culture, a 4% (w / v) milky solution of "soluble starch" from Acros Organics (Division of Thermo Fisher Scientific, Geel, Belgium) was produced. -1 Maltose basal medium (maltose·1H2O). Media with different pH and osmotic pressure were generated by adding correctors (HCl or NaOH) and by changing the concentration of the carbon source maltose, respectively, by adding inositol, which according to our studies cannot be metabolized (data not shown).

[0400] In addition, a 1 g·L -1 , 2g·L -1 , 3g·L -1 , 4g·L -1 and 5g·L -1A "soluble starch" based medium for cultivation under limited carbon source.

[0401] The pH and osmolarity of all media were determined by means of a pH meter Calimatic from Knick GmbH (Berlin, Germany) and an Osmomat 3000 from Gonotec GmbH (Berlin, Germany) according to the manufacturers' instructions.

[0402] Shake flask culture

[0403] In a GFL shaking incubator 3032 or 3033 (Burgwedel, Germany), 250 mL of The culture was carried out for seven days in Erlenmeyer baffled cell culture flasks at 28°C and 140 rpm. For inoculation of 50 mL of culture medium, 1 mL of spore solution with OD = 3-5 was used. The dry cell weight was determined as described by Wolf et al. (2017a). The supernatant was stored at -20°C for subsequent analysis.

[0404] Microculture in the BioLector system of m2p-labs GmbH (Baesweiler, Germany)

[0405] Comparative growth experiments were performed in 1 mL reaction volumes in 48-well FlowerPlate (m2p-Labs GmbH, Baesweiler, Germany) covered with gas-permeable sealing foil and in the presence of The cells were cultured at 28°C and 800 rpm for 1 week. Growth was recorded by backscattering signals. To determine the final cell dry weight, 800 μL / well was sampled in a weighed reaction tube (14,000 g, 2 minutes), washed with deionized water, and dried at 60-70°C for 1 day. The supernatant was stored at -20°C for subsequent analysis.

[0406] Recombinant DNA research

[0407] Unless otherwise stated, plasmid construction and assembly were performed by Gibson assembly (Gibson et al. 2009). Fragments were amplified using High-Fidelity PCR Master Mix with GC Buffer (NEB, Ipswich, MA, USA) and, when necessary, treated with DpnI (Thermo Fisher Scientific, Waltham, MA, USA). PCR products and gel extracts were purified by using Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). Equimolar amounts of DNA fragments were added to Gibson Assembly Master Mix at a ratio of 1:4. The master mix consisted of 0.64 μL T5 Exonuclease (10 U μL -1 , NEB, Ipswich, MA, USA), 20 μL Phusion High-Fidelity DNA Polymerase (2U·μL -1 , Thermo Fisher Scientific, US), 160 μL Taq DNA Ligase (NEB, Ipswich, MA, USA), 699.36 μL distilled water and 320 μL isothermal reaction buffer (25% PEG-8000, 1 mL 1M Tris-HCl, 100 μL 1M MgCl2, 100 μL 1M DTT, 20 μL of each 1mM dNTP, 200 μL NAD). The samples were incubated at 50 °C for at least 1 hour and then transferred to Escherichia coli DH5αMCR by chemical transformation according to the protocol of Beyer et al (2015). In the presence of 15 g·L -1 Agar (Carl Roth, GmbH & Co, Karlsruhe, Germany) and 50 mg L -1 Selection for E. coli was performed on Luria / Miller broth containing apramycin sulfate. Positive colonies were tested by PCR and gel electrophoresis and by Sanger sequencing by an in-house sequencing core.

[0408] Construction of plasmid for gusA reporter system

[0409] For the construction of the plasmid of the gusA reporter system, see Schaffert et al. (2019).

[0410] Construction of a new pSETT4 expression system

[0411] To clone the new pSETT4 expression system, the pSET152 vector of Bierman et al. (1992) was used as template. The vector backbone was linearized by PCR (Table E5).

[0412] The cloning cassette consisting of the gapDH promoter, the lacZ gene under the control of the lac-promoter and several restriction sites of three T4 terminators on the sides was ordered as string DNA at Integrated DNA Technologies (Iowa, USA). Due to the complex structure, the cassette was ordered in three parts and assembled using the primers in Table E5 by GeneSowing (Horton 1995). Finally, the backbone and insert were assembled by Gibson Assembly (Gibson et al. 2009). The new vector system was named pSETT4gap.

[0413] To exchange 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 primers tipA_GAF and tipA_GAR and assembled with the linearized backbone by Gibson assembly (Gibson et al. 2009). This vector was named pSETT4tip (see Figure 6 ).

[0414] Table E5 Gibson assembly primers for assembly of the new expression systems pSETT4gap and pSETT4tip

[0415]

[0416] Overexpression of single genes in the novel pSETT4 expression system

[0417] For overexpression of individual genes, inserts were 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). To express the acbB gene under the control of the native promoter, the vector backbone pSETT4gap was digested with BsaI and NdeI, resulting in linearization of the vector after 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).

[0418] Table E6 Primers for Golden Gate cloning and restriction cloning into insert amplification in pSETT4gap and pSETT4tip vector systems

[0419]

[0420] Construction of pCRISPomyces-2 deletion and integration vectors

[0421] For the 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 spacers and their reverse complements were ordered as oligonucleotides with overlap at metabion GmbH (Steinkirchen, Germany) or Sigma-Aldrich (Taufkirchen, Germany) (Table E7).

[0422] According to the protocol of Cobb et al. (2015), the oligonucleotides were annealed into double strands and assembled with plasmids by Golden Gate Assembly (Engler et al. 2008). To repair Cas9-induced double-strand breaks, the DNA template was cloned into the vector backbone by Gibson Assembly (Gibson et al. 2009). As DNA templates, flanking sequences (approximately 1 kB) upstream and downstream of the target gene were amplified from genomic DNA by PCR (Table E8).

[0423] Table E7 Spacers and reverse complements used in Golden Gate Assembly with pCRISPomyes-2.

[0424]

[0425] Table E8 Gibson Assembly primers for pCRISPomyes-2 deletion and integration vectors.

[0426]

[0427] Deletion of the gene cgt by CRISPR / Cas9 technology

[0428] To construct the Δcgt (ΔACSP50_5024) deletion mutant by CRISPR / Cas9 technology (clustered regularly interspaced short palindromic repeats / CRISPR-associated endonuclease 9), the plasmid pCRISPomyces-2 (Cobb et al. 2015) was used. The spacer sequences were selected according to Wolf et al. (2016) and ordered as oligonucleotides together with their reverse complement at metabion GmbH (Steinkirchen, Germany) (spacer_1: 5'-acgcAGCGTCGCCCGCTGGGAGAA-3', spacer_2: 5'-aaacTTCTCCCAGCGGGCGACGCT-3'). The oligonucleotides were annealed into duplexes and assembled with the plasmid by GoldenGate Assembly (Engler et al. 2008) using BsaI (NEB, Ipswich, MA, USA) according to the protocol of Cobb et al. (2015) (Cobb et al. 2015). To repair Cas9-induced double-strand breaks, deoxyribonucleic acid (DNA) templates were cloned into XbaI-linearized vectors by Gibson Assembly (Gibson et al. 2009). High-Fidelity PCR Master Mix with GC Buffer (NEB, Ipswich, MA, USA) (primer sequences: cgt_flank1_fw: 5′-tcggttgccgccgggcgttttttatCCGGTACCCTGCTCCTCGTC-3′, cgt_flank1_rv: 5′-gtgacgcattgacgcaggtcGAGGGATATGGCTCAGATAC-3′, cgt_flank2_fw: 5′-gtatctgagccatatccctcGACCTGCGTCAATGCGTCAC-3′, cgt_flank2_rv: 5′-gcggcctttttacggttcctggcctACCTGACCCTGCTGAAATGG-3′) was used to amplify the flanking sequences (approximately 1 kB) upstream and downstream of the target gene by polymerase chain reaction. For Gibson Assembly, an equimolar mixture of DNA fragments (Flank_1: 1101 bp and Flank_2: 982 bp) was added to Gibson Assembly Master Mix at a ratio of 1:4, which consisted 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 1M Tris-HCl, 100 μL of 1M MgCl2, 100 μL of 1M DTT, 20 μL of each 1 mM dNTP, 200 μL of NAD). After incubation at 50°C for at least 1 hour, the reaction mixture was transferred to E. coli DH5αMCR by chemical transformation according to the protocol of (Beyeret al. 2015). -1 15 g L of Apramycin Sulfate -1Growth and selection of E. coli were performed on agar Kobel's Luria / Miller broth (LB-medium) (both: Carl Roth, GmbH & Co KG, Karlsruhe, Germany). Plates were incubated at 37°C for 10-14 hours. Apramycin-resistant colonies were first tested by PCR and gel electrophoresis followed by Sanger sequencing by our in-house sequencing core facility (primer sequences for PCR: 5'-GGCGTTCCTGCAATTCTTAG-3', rev: 5'-TCGCCACCTCTGACTTGAGC-3', walking primers for sequencing: w1: 5'-CGCTGATCTTCAGCTTCC-3', w2: 5'-GCCTTCACCTTCCATCTG-3', w3: 5'-TCGGGAAAGCCGCCGGAG-3').

[0429] Actinomycetes SE50 / 110

[0430] Competent Actinomycetes SE50 / 110 cells were prepared from freshly grown NBS cultures (see above). The cells were washed twice in 10% (w / v) ice-cold sucrose and twice in ice-cold 15% (v / v) glycerol. Finally, the cells were collected in 15% (v / v) ice-cold glycerol (by adding approximately four times the volume of the cell pellet), aliquoted to 100 μL in a reaction tube, and quickly frozen in liquid nitrogen. The competent Actinomycetes cells were stored at -80°C.

[0431] For conjugation, E. coli ET12567 / pUZ8002 (Kieser et al., 2000) was used. The desired construct was transferred into E. coli ET12567 / pUZ8002 according to Beyer et al. (2015) and supplemented with 50 mg·L -1 Apramycin sulfate, 50 mg·L -1 Kanamycin sulfate and 15 mg·L -1 After selection on LB agar plates of chloramphenicol, cells were grown in liquid culture medium (LB medium with the same supplements) and harvested at an optical density of 0.4-0.6. Cells were washed twice in ice-cold LB medium and mixed with competent actinomycetes SE50 / 110 cells. Cell suspensions were plated on SFM agar plates. After culturing for 20-24 hours at 28°C, 1 mL was dissolved in 500 mg L in ddH2O. -1 Apramycin sulfate was spread on the plate with a sterile swab. After 1 week, the first post-conjugative bodies of Actinomycetes SE50 / 110 could be observed. The post-conjugative bodies were transferred to a plate supplemented with 50 mg·L -1SFM agar plates containing apramycin sulfate. Repeated streaking is performed several times to purify the post-conjugative body of actinomycetes from E. coli. To accelerate the process, the medium can be supplemented with 50 mg·L -1 Fosfomycin or trimethoprim to remove the donor strain.

[0432] Plasmid elimination to obtain marker-free CRISPR / Cas9 deletion / integration mutants of Actinomycetes SE50 / 110

[0433] Plasmid elimination was performed by culturing in complex medium NBS at elevated temperatures according to the protocol of Wolf et al. (2016). The presence of plasmids in colonies was tested by streaking in parallel on SFM plates containing and without apramycin. The absence of apramycin-sensitive postconjugation bodies was tested by PCR (primer sequence data not shown). PCR fragments were cut from the gel and sequenced by our in-house Sanger sequencing core facility.

[0434] In addition, genomic DNA of deletion or integration mutants was sequenced by Oxford Nanopore technology (Oxford, UK) to exclude off-target effects. Genomic DNA from NBS grown cultures was isolated using the Microbial DNA Kit (Macherey-Nagel, Düren, Germany). Libraries were prepared with the aid of ID genomic DNA by ligation kit (Oxford Nanopore, Oxford, UK).

[0435] Deletion system based on homologous recombination and counterselection with the cytosine deaminase codA

[0436] By using the replicative vector pKC1139, the vector was integrated into the genes of the acb gene cluster. Based on this observation, a new deletion system using homologous recombination was developed and tested by the example of the gene cgt (ACSP50_5024).

[0437] A vector backbone with an origin of transfer (ncP) and the relaxosome gene traJ was used to allow conjugation into A. apramycin SE50 / 110. In this study, two different antibiotic resistance markers mediating apramycin and kanamycin resistance were tested for selection: aph(3')II (kan R , kanamycin) and aac(3)IV (apm R , apramycin). In addition, a high copy number ColE1 / pMB1 / pBR322 / pUC replication origin was integrated to allow replication in the donor strain E. coli. ori, oriT ncPThe tra gene and resistance cassette were taken from pRT802 and pRT801 (Gregory et al. 2003), respectively. Since the new deletion system does not contain a replicon for replication in Actinomycetes SE50 / 110, nor does it contain an integrase gene with a junction site, when integrated into the genome by homologous recombination, the vector can only be maintained in Actinomycetes SE50 / 110 ( Figure 7 ). For this reason, a 2kB homologous sequence was integrated, which is located at the flank of gene cgt. After conjugation transfer in Actinomycetes SE 50 / 110, the mutant in which the first exchange has occurred can be selected by apramycin or kanamycin resistance. In order to force the carrier backbone to integrate (the second exchange), 5-fluorocytosine (5-FC) is added, which is converted into toxic product 5-fluorouracil (5-FU) by cytosine deaminase CodA. In this study, codA (s) is used, which is codon-optimized (Dubeau et al., 2009) for Streptomyces species (Streptomyces ssp.). After the second exchange, there is a genotype of the wild type or a genotype of the deletion mutant.

[0438] The new deletion system was successfully tested for the gene cgt, as shown by colony PCR and ONT sequencing. After a successful second crossover, the proportion of deletion mutants was between 25% and 32%. The workflow is shown in Figure 8 middle.

[0439] Analytical methods

[0440] Quantification of acarbose from the supernatant by HPLC

[0441] The supernatant of the maltose-grown culture of Actinomycetes was centrifuged (20,000 g, 2 min), mixed with methanol 1:5 by vortexing, and centrifuged again to remove the precipitate (20,000 g, 2 min). The samples were transferred to HPLC vials and analyzed in an Agilent HPLC system 1100 series (G1312A binary pump Serial#_DE43616357, G1329A ALS autosampler Serial#_DE43613 / 10, G1315A diode array detector (DAD) Serial#_DE72002469). A Hypersil APS-2 column (125×4 mm, 3 μm particle size) from Thermo Fisher Scientific Inc. (Waltham, MA, USA) was used as the stationary phase and heated to 40°C. 1 mL·min was used. -1 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 used as the mobile phase. 40 μL of each sample was injected and separated in a 10-minute run. Acarbose was detected with a DAD detector at 210 nm (reference 360 ​​nm) and quantified from the peak area of ​​the calibration curve.

[0442] Liquid chromatography-mass spectrometry (LC-MS)

[0443] Sample preparation for analysis of intracellular metabolites

[0444] Triplicate A. aeruginosa SE50 / 110 strains were grown in maltose basal medium for at least 4 days. 10 mL of the culture was rapidly filtered through filter paper using a Buchner funnel and washed with 2.63 g·L -1 The cells were washed with NaCl solution. The cells were transferred to pre-weighed round-bottom screw cap tubes, snap frozen in liquid nitrogen, and stored at -80 °C. The cells were dried overnight in a centrifugal evaporator (SpeedVac) of ThermoFisher Scientific (Waltham, MA, USA). The 4 mg dried cells were transferred to a new 2 mL screw cap tube containing a mixture of about 500 μL of zirconium oxide / silicon dioxide microbeads (Bio Spec Products Inc., Bartlesville, USA) with a size of 0.1 mm, 0.05 mm, and 0.01 mm. 700 μL of 80% MeOH was added to the cells and beads. Cells were broken three times for 30 s at a speed of 6.5 in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA). The samples were cooled on ice for 5 minutes during this 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 stream of nitrogen, and dissolved in 50 μL of distilled water.

[0445] Sample preparation for analysis of extracellular Akavirid metabolites

[0446] Sample preparation was performed according to the protocol described by Ortseifen (2016). Sugars and pseudosugars were enriched from 10 mL of supernatant by solid phase extraction using Easy columns (Macherey-Nagel, Düren, 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 in 3 mL of methanol.

[0447] LC-ESI-MS of intracellular and extracellular metabolites

[0448] For LC-MS, a Lachromultra (Hitachi Europe Ltd., UK) HPLC system coupled to a MicroTOF-Q hybrid quadrupole / time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) source was used.

[0449] To analyze intracellular metabolites, 2 μL of sample was separated on a pHILIC 5 μm Polymeric column (150×2.1 mm) (Merck, Darmstadt, Germany). The following gradient was used at 0.2 mL min -1 Eluent A (20 mM NH4HCO3, pH 9.3, adjusted with aqueous ammonia solution) and eluent B (acetonitrile) were used at flow rates of: 0 min B: 90%, 30 min B: 25%, 37.5 min B: 25%, 40.0 min B: 80%.

[0450] As a standard for peak identification, 2 μL of 10 μM UDP-glucose, glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, and dTDP-glucose were injected.

[0451] To analyze extracellular Akavirid metabolites, Cogent Diamond Hydride TM 10 μL of the sample was separated on an HPLC column (Microsolv Technology Corporation; 150 mm×2.1 mm; 3 μL particle size). The following gradient was used at 0.4 mL min -1 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 used at flow rates: 0 min B: 100%, 8 min B: 0%, 13 min B: 0%, 15.5 min B: 100%, 18 min B: 100%.

[0452] The ESI source was operated in negative ionization mode for analyzing intracellular metabolites and in positive ionization mode for analyzing extracellular Akavidin metabolites. The temperature of the drying gas and capillary was set to 180° C. The scan ranges of MS were set to 200-1,000 m / z (intracellular metabolites) and 50-3,000 m / z (extracellular Akavidin metabolites), respectively.

[0453] By using the software CompassTM The mass-specific peak areas were integrated using a PCR amplification assay (Bruker Daltonics, Bremen, Germany). Peaks were normalized to the weighed weight of dried cells (intracellular metabolites) and to the dry weight of cells at the time of sampling (extracellular akavidin metabolites).

[0454] Extraction and analysis of carotenoids

[0455] extract

[0456] Cell pellets from Actinomycetes SE50 / 110 were transferred to 2 mL screw cap tubes with approximately 500 μL of a mixture of 0.1 mm, 0.05 mm and 0.01 mm zirconium oxide / silica microbeads (Bio SpecProducts Inc., Bartlesville, USA). 1 mL of acetone or methanol was added as the extraction solvent. Cell disruption was performed three times for 45 s 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 min in between. 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 acetone extract and methanol extract was produced at a ratio of 7: 3 and transferred to a new glass vial.

[0457] Thin layer chromatography (TLC) and spectroscopic analysis

[0458] The carotenoids extracted by 50 μ L are applied to a silica gel matrix (HPTLC-HL, catalog number 58077, Analtech Inc., Newark, USA) with 5 μ L steps, and incubated in a TLC chamber filled with 100 mL petroleum, 11 mL isopropanol and 50 μ L water. Run in the dark. After the TLC plate is dried, the band is peeled off with a scalpel and transferred to a new tube. After adding 1 mL of ethanol, the absorption spectrum is analyzed by using the Genesys 10S UV-Vis spectrophotometer of Thermo Fisher Scientific (Waltham, MA, USA).

[0459] HPLC Analysis of Carotenoids Using Absorbance Scanning

[0460] Carotenoids were separated by reverse-phase HPLC according to Henke et al. (2017) and Heider et al. (2014) using an Agilent 1200 series HPLC system (Agilent Technologies GmbH & Co. KG, Germany) including a diode array detector (DAD) for UV-Vis spectroscopy. A sample volume of 20 μL was added at 0.5 mL min -1 A precolumn (10 × 4 mm MultoHigh 100RP18-5) and a main column (ProntoSIL 200-5C30, 250 × 4 mm) from CS ChromatographieService GmbH (Langerwehe, Germany) were used as stationary phases as described previously (Heider et al. 2014; Henke et al. 2017).

[0461] 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 a 0.1 M ammonium acetate solution 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 the run.

[0462] Determination

[0463] Promoter screening assay by spectrophotometric measurement of glucuronidase activity

[0464] Two different types of glucuronidase assays were performed: one with crude protein extracts and one with intact cells. The protocols described by Horbal et al. (2013) and Siegl et al. (2013) were adapted to A. planarians SE50 / 110. The substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc, AppliChem GmbH, Darmstadt, Germany) was chosen because the substrate p-nitrophenyl-D-glucuronide dissociates under our assay conditions.

[0465] Growth conditions and sample preparation

[0466] As mentioned above, the mutant of actinomycetes carrying the promoter construct with gusA gene was cultivated in maltose basal medium for one week.Measured during the growth period.Sampling 500 μ L of each culture was used for the determination of intact cells.Sampling 1 ml was used for the determination of crude protein extract, and transferred to a screw cap tube containing zirconium oxide / silicon dioxide microbeads (Bio Spec Products Inc., Bartlesville, USA) with a size of 0.1 mm and 0.05 mm.Cells were broken twice for 30 seconds and 5 minutes in the middle on ice in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) with a speed setting of 6.5.After centrifugation, the lysate was transferred to a new reaction tube and centrifuged.Supernatant was used for cell-free determination.Total protein was quantified by Bradford determination (see above).

[0467] Glucuronidase (GUS) assay

[0468] In black microtiter plates (96-well PS F-bottom μCLEAR, black, medium binding, Greiner Bio-One, Kremsmünster, GUS assay was performed in REF 655096. 100 μL of each sample (cell suspension or lysate) was pipetted into three wells, one of which was used as a negative control and two as technical replicates. GUS buffer (50 mM phosphate buffer pH 7.0 (5.136 g L -1 Na2HPO4·2H2O, 3.299 g·L -1 Na2PO4·2H2O) containing 5mM DTT and 0.1% Triton-X-100) supplemented with 2mM substrate X-Gluc (stock solution: 0.2M in DMF). 100 μL was added to 100 μL of sample. For negative control, 100 μL of gus buffer without substrate was added. In addition to separate negative controls for each sample, medium and substrate controls were also prepared.

[0469] In a preheated Tecan Reader Infinite M200 (Ref 30016056, Tecan Group AG, Switzerland) (37 ° C) for 3 hours (measured with intact cells) and 2 hours (measured with lysates). The maximum absorption of indigo was measured at 610 and 660 nm. After deducting the absorption values ​​of all controls, the slope of each absorption curve was calculated by linear regression and normalized to the cell dry weight (measured with intact cells) or to the total protein amount (measured with lysates). The normalized slope was used to compare the β-glucuronidase activity in different mutants.

[0470] Screening experiments in the OmniLog phenotypic microarray system

[0471] exist Prescreening experiments were performed in the OmniLog Identification System (Hayward, CA, USA) to evaluate respiration on different carbon sources (PM1 and PM2 plates). Actinomycetes SE50 / 110 wild type and deletion mutant Δcgt were grown on SFM agar plates as described elsewhere herein. Cells were harvested by using a sterile swab and diluted in inoculum IF-0a of PM1 and PM2. The turbidity of the cell suspension was checked in a turbidimeter to achieve 80% transmittance. According to the manufacturer's protocol, 2.32 ml of 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 1.5 M NH4Cl, 0.24 ml of 1.0 M Na3PO4, 0.24 ml of distilled water, 0.24 ml of Biolog redox dye mixture G and 0.24 ml of metal ion mixture (5.0 mM each: ZnCl2 7H2O, FeCl2 6H2O, MnCl2 4H2O, CaCl2 2H2O). PM plates were inoculated with 100 μL / well of the prepared solution and cultured for 1 week at 28-30°C in an OmniLog system (model 71000Serial#_406). Data evaluation was performed using the manufacturer's software (KineticAnalysis, Biolog and Omnilog 2.3, Biolog).

[0472] RNA research

[0473] Sampling and RNA isolation

[0474] For transcriptome analysis, 2 × 1 mL of culture 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.

[0475] To isolate RNA, the frozen cell pellet was resuspended in 500 μL of LB buffer ( RNA Plus, Macherey-Nagel, Düren, 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 in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) at speed setting 6.5 three times for 20 seconds and 5 minutes on ice in between. Subsequently, the cell suspension was centrifuged at 13,000 g and 4 ° C for 5 minutes. Use The RNA Plus kit was combined with the rDNase Set (Macherey-Nagel, Düren, Germany) for on-column DNA digestion and the supernatant was used for RNA extraction. After purification (clean-up) and elution according to the manufacturer's protocol, the DNA digestion was repeated (in solution) and the sample was purified again using the same kit. The samples were tested for residual DNA using two primer pairs binding to the genomic DNA of Actinomycetes SE50 / 110 and amplification of small fragments of approximately 200-300nt. If necessary, the DNA digestion and RNA purification were repeated. The amount of RNA was analyzed using a NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany).

[0476] Reverse transcription quantitative PCR

[0477] Reverse transcription quantitative PCR was performed in the Roche LightCycler 96 system (Mannheim, Germany) using the SensiFast SYBR No-Rox One-Step Kit (Bioline, London, UK) and 96-well lightcycler plates (Sarstedt, Nümbrecht, Germany) according to the protocol of Wolf et al. (2017a). Relative RNA amounts were normalized to total RNA (100 ng) and calculated as 2 -ΔCq ΔCq is the difference in the average Cq of the mutant strain compared to the control strain. The primers in Table E9 were used to determine the relative transcription of the genes.

[0478] Table E9 Primers used in RT-qPCR experiments

[0479]

[0480]

[0481] Whole genome oligonucleotide microarray

[0482] Whole-genome oligonucleotide microarrays were performed according to the protocol of Wolf et al. (2017a), which adapted the hybridization method to the high G+C content of A. planaris SE50 / 110.

[0483] RNA was isolated in triplicate and pooled in equimolar amounts (total amount of 5 μg pooled RNA in 12 μL). For cDNA synthesis, labeling, and microarray hybridization, the Two-Color Microarray-Based ProkaryoteAnalysis FairPlay III Labeling Kit (version 1.4, Agilent Technologies, Santa Clara, CA, USA) was used according to the manufacturer's instructions with practical adjustments as described by Wolf et al. (2017a). Labeling was performed using Amersham CyDye single reactive dye packages (GE Healthcare, Little Chalfont, UK). A custom whole-genome oligonucleotide microarray representing the sequence of Actinomycetes SE50 / 110 was used, which was designed by Wolf et al. (2017a) (4×44K format, 43,803 features representing 8,238 genes and 1,417 control points, supplier: Agilent Technologies, Santa Clara, CA, USA). All microarray-specific reagents and devices from Agilent Technologies (Santa Clara, CA, USA), including hybridization ovens and scanners, were used. Feature extraction was performed using Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies, Santa Clara, CA, USA) (Protocol GE2_107_Sep09). Subsequent data analysis, including LOWESS normalization and statistical analysis, was performed using the microarray and gene expression (MAGE)-compatible system EMMA 2 (Dondrup et al. 2009). A p-value of 0.05 was used as the cutoff for significance. According to the "yellow experiment" previously performed by Wolf et al. (2017a), the M-value cutoffs for a false discovery rate of 0.01 were determined to be 1.1 and -1.1.

[0484] Analysis of functional relevance of Cgt

[0485] Distribution of single-domain CBM-20 proteins in the eubacteria kingdom

[0486] By BlastP analysis, the present inventors analyzed the distribution of CBM-20 single domain protein in the prokaryotic kingdom.

[0487] Briefly, the distribution of single 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 a variety of different proteins and enzymes, data filtering was necessary: ​​of the initial 3,316 BlastP hits, all eukaryotic origins and all enzymes with functional specific annotations or sizes greater than 350 amino acids were excluded. The remaining 80 BlastP hits were analyzed for domain structure (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, contained two CBM-20 domains traversed by a higher-order domain known as glyco-hydro-77-superfamily 4-α-glucanotransferase. Ten contained different additional domains: five α-amylase inhibitor domains, two CBM-25, N-terminal CBM-26 binding domains, two N-terminal domains of the IPT superfamily with possible regulatory functions, and a DUF1393 domain, which was described to be present in several α-amylases (information taken from the NCBI database). These candidates were also excluded. Only 18 candidates (including Cgt from Actinomycetes SE50 / 110) showed a single CBM-20 domain. A protein tree was established by Blast Tree View 1.17.5 of the NCBI database (NCBI database), based on a multiple sequence alignment performed by BlastP (Altschul et al. 1990; Altschul et al. 2005).

[0488] Interestingly, single CBM-20 domain proteins were found in only 17 other species ( Fig. 9). Most of these are found in species of the order Actinomycetales, for example in all strains of the genus Actinomyces. Most of the 17 species were originally isolated from soil and environmental samples, namely A. missouriensis (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 OK885 (isolated from roots, Tennessee, USA, information taken from GenBank (Benson et al. 2013) of NCBI (NCBI database)), Streptosporangium roseum (Nolan et al. 2010), Streptosporangium sclerotialus (synonym Chainia antibiotica) (Thirumalachar 1955), Cellulomonas sp. B6 (Piccinni et al. 2016), Paenibacillus sp. P22 (Hanak et al. 2014), and Clostridium sp. DMHC 10, which was isolated from sludge in a winery waste treatment unit (Kamalaskar et al. 2010). CBM-20 proteins are also present in Streptomyces DI166 (whose sampling site has not been reported) and multiple species of the Pseudomonadaceae family. They belong to genera known to include soil-inhabiting members.

[0489] Strains carrying a single CBM-20 protein that is not directly associated with the soil or environment occur only occasionally, such as in single isolates of the human pathogens Chlamydia trachomatis (Thomson et al. 2008) and Mycobacterium abscessus (Ryan and Byrd 2018; Moore and Frerichs 1953).

[0490] Confirmation of starch binding function by in vitro assay

[0491] The CBM-20 domain has been described as having a starch binding function, and the inventors wish to test this function by in vitro assays. Since the N-terminal signal peptide causes the small carbohydrate binding protein Cgt to be highly expressed and enriched in the extracellular space (Wendler et al. 2015a), the protein can be concentrated directly from the supernatant by filtration. Starch binding analysis was performed with potato starch at different concentrations. Both the starch fraction and the supernatant were analyzed by SDS-PAGE. In all starch fractions (1-10% (w / v) of starch), a protein band at about 15 kDA was detected, which was clearly identified as Cgt by MALDI-TOF-MS. In contrast, the supernatant fraction was almost completely depleted by Cgt. Residual Cgt was found in the supernatant, indicating that the added starch was completely saturated with Cgt. In the negative control without starch, most of Cgt remained in the supernatant fraction. In addition to Cgt, another small extracellular protein ACSP50_6253 of unknown function was identified by starch binding analysis (data not shown).

[0492] Analysis of cgt expression during growth on different carbon sources

[0493] It has been reported that the gene cgt is differentially expressed in the presence of different carbon sources, as determined by transcriptome and proteome analysis on glucose and maltose (Schwientek et al., 2013; Wendler et al., 2015a; Ortseifen 2016). The present inventors tested the effect of several carbon sources on the expression of the cgt gene by measuring the amount of transcripts by reverse transcription quantitative PCR (RT-qPCR). For this purpose, the wild-type strain of Actinomycetes SE50 / 110 was grown on a basal medium supplemented with maltose, glucose, starch, galactose, cellobiose, lactose and C-Pur (Cerestar 01908) ( Fig.10 ). The latter is a sugary product from starch degradation, consisting mainly of maltose and maltotriose. All carbon sources were supplemented with equal C-molar amounts. The only exception was starch: due to its low solubility, a 4% (w / v) milky white solution of "soluble starch" from Acros Organics was produced here. For comparison, a maltose-based medium with reduced maltose content was prepared (here: 44.40 g L -1 Maltose monohydrate), where the C molar concentration should be close to the C molar concentration in the starch medium.

[0494] For most carbon sources tested, transcription of the cgt gene was similar or only slightly and not significantly reduced compared to maltose-grown cultures ( Fig.11A). A lesser degree of differential transcription was observed for galactose (3.4 fewer transcripts, log2(fold change) = 0.291). A significant reduction in cgt transcripts was determined for the carbon sources glucose (142-fold reduction in transcription, log2(fold change) = 0.007) and lactose (62-fold reduction in transcription, log2(fold change) = 0.016). When cells were grown in maltose-based medium with reduced amounts of maltose (here: 44.4 g L -1 , instead of 72.06 g·L -1 ) was observed to decrease the transcription of the cgt gene by 2.9-fold (log2(fold change)=0.345)( Fig.11 B).

[0495] Analysis of the gene deletion mutant Δcgt

[0496] Δcgt on different carbon sources or under carbon-limiting conditions

[0497] The differential transcriptional profile of Cgt in relation to carbon source suggests a function in sugar metabolism, as previously thought (Ortseifen 2016). Ortseifen (2016) (Ortseifen 2016) suggested that Cgt is responsible for retaining carbon as an energy source in the context of the Carbophore model. The growth of wild type and CRISPR / Cas9 deletion mutant Δcgt was tested on different carbon sources in liquid culture.

[0498] Previously, pre-screening experiments were performed in the OmniLog phenotypic microarray system (Biolog Inc., Hayward, United States of America), which allows rapid phenotypic screening by measuring cellular respiration activity on a total of 190 different carbon sources in a multiwell plate. Among them, actinomycetes showed respiration on 103 carbon sources. Except for arabinose and lactose, no differential respiration characteristics were observed for Δcgt on the remaining 101 carbon sources. In order to verify these results at the growth level, the carbon sources arabinose and lactose were further tested in shake flask cultures. In addition, standard laboratory sugars maltose and glucose, and complex carbon sources starch and the disaccharide cellobiose were tested to simulate the natural carbon sources of habitat soil. No restriction to growth was observed for Δcgt ( Fig.12 and Fig.13 ).

[0499] In addition, at m2p-labs - The system was tested under carbon-limited conditions (here: 1 g·L -1 , 2g·L -1 , 3g L -1 4g L -1 and 5g·L -1Compared with the wild type, no growth disadvantage of the Δcgt mutant was observed under carbon source limitation ( Fig.14 ).

[0500] Δcgt has no effect on osmotic pressure tolerance or pH tolerance

[0501] Cgt polymers have been suggested to form a surface layer by polymerizing (Wendler et al. 2015a). This may indicate a potential role in protection against environmental changes such as drought, pH and osmotic stress.

[0502] exist pH screening was performed on solid medium in the ATCC-1700 system as well as in liquid culture. For screening on solid medium, SFM-agar plates with a pH range from pH 4 to 11 were prepared (in step 1) and drops of serial dilutions of spores of wild type and deletion mutant Δcgt were applied. Both the mutant and wild type were able to grow from pH 5 to 11. No differences in growth or spore formation were observed on the agar plates.

[0503] Since the effect of drought tolerance is difficult to assess, the present inventors analyzed colony formation and spore formation on the surface of bacterial lawns and found no difference between wild type and Δcgt.

[0504] For pH screening in liquid culture, maltose-based media was prepared with a pH range of 4 to 7. Higher pH values ​​could not be tested in liquid culture because media components tended to precipitate. Both strains were grown from pH 4.5 to 7 ( Fig.15 ). No differences were observed regarding final dry cell weight.

[0505] For osmotic pressure screening, use 3.6 to 108.1 g·L -1 The different maltose concentrations of maltose monohydrate ranged from 323.5 to 681.0 mOsmol kg -1 Maltose-based medium was prepared with a range of osmotic pressures (Table E11). No significant growth difference was observed between the wild type and the deletion mutant Δcgt ( Fig.16 ).

[0506] In addition, inositol was tested as an osmotic agent because it is not consumed by actinomycetes. Here, osmolarity ranged from 388.5 to 695.0 mOsmol kg -1 , but no growth differences were observed ( Fig.17 ).

[0507] By using the complex medium NBS, the test was performed at 159-190 mOsmol kg -1Furthermore, no significant growth difference was observed between the wild-type strain and the deletion mutant Δcgt.

[0508] Table E10 Tabular summary of the screening experiment. Final cell dry weight, final acarbose concentration, pH and osmolarity of different basal media used for screening osmolarity and pH in this study. The different osmolarities in the media used for pH screening were introduced by the addition of a corrector.

[0509]

[0510]

[0511] Δcgt shows improved acarbose formation on maltose-based medium

[0512] Although no obvious growth phenotype was observed under the conditions tested, the lack of highly expressed Cgt protein appears to save metabolic resources of the cell, such as ATP and amino acids. These can be used for cell growth or other anabolic processes. In the experiments, Δcgt did not show a significant growth advantage. However, significantly higher final acarbose concentrations were detected for the deletion mutant Δcgt compared to the wild type (Table E10). For cultures in complex medium, this was most pronounced during the growth phase ( Fig.18 ).

[0513] The improvement of the acarbose-producing phenotype was confirmed by three independent shake flask cultures in maltose-basal medium ( Fig.19 and Table E11). Quantification of acarbose from the supernatant showed an increase in the acarbose yield coefficient of the deletion mutant compared to the wild type. The difference in the final acarbose yield was significant (tested by a two-sided t-test, p value = 0.04608). Thus, in Δcgt, an increase of 8.3% to 16.6% in the final acarbose concentration was achieved (see Table E11).

[0514] Table E11. Tabular summary of the growth experiment under acarbose production conditions. Final acarbose concentration, final dry cell weight and number of replicates n for three independent cultures of Δcgt and wild type in maltose basal medium.

[0515]

[0516] Δcgt had no effect on the expression of acarbose biosynthesis genes

[0517] It was surprising to find that the deletion of the highly expressed gene cgt had no negative effect on the growth or viability of the organism under various conditions, but produced an enhanced acarbose production phenotype. Due to this, and in order to exclude a direct effect on the regulation of acarbose biosynthesis (acb) genes, RT-qPCR of representative acb genes was performed. To this end, wild type and Δcgt were grown on maltose basal medium and RNA was isolated from samples of the early growth phase. The relative transcript amounts of the acarbose biosynthesis cluster genes acbZ, acbW, acbV, acbA, acbB, acbD and acbE were calculated for Δcgt compared to wild type ( Fig. 20 ). Gene acbV is the first of several polycistronically transcribed genes within the main operon of the acarbose biosynthetic gene cluster (Wolf et al. 2017b). The monocistronic transcribed genes acbD and acbE encoding extracellular acarbose metabolic proteins have been shown to be strongly regulated by the acarbose regulator AcrC (Wolf et al. 2017a). Genes acbA, acbB and acbZ are also monocistronic and annotated as enzymes for acarbose biosynthesis (acbAB) and its extracellular metabolism (acbZ), respectively. AcbW is the first gene of the acbWXY operon and is putatively encoding an ABC transporter. For all selected transcripts, no significant changes in relative transcript levels were measured in the deletion mutant Δcgt compared to the wild type ( Fig. 20 ).

[0518] discuss

[0519] The relationship between carbohydrate metabolism and acarbose biosynthesis is of high interest. Recent studies pointed out the importance of carbon utilization in the context of the biosynthesis of acarbose and further acarbose metabolites in the wild type (Wendler et al., 2014).

[0520] In this case, the starch binding protein Cgt is significant. It is one of the most strongly expressed genes in Actinomycetes SE50 / 110 (Schwientek et al. 2013), accounting for about 8% of the entire secretory proteome (unpublished data of the inventors). Its gene product is exported to the extracellular space (Wendler et al. 2013). Overproduction and export means high costs for cells: 4 ATPs are required for each peptide bond for the translation process alone (Campbell and Reee 2011; Purves 2006), i.e., not including the additional costs of RNA synthesis, amino acid production, protein folding and export. Therefore, the inventors conclude that Cgt plays an important role in the physiology of Actinomycetes SE50 / 110. Two different functions of Cgt are proposed and analyzed herein: a role in sugar metabolism and a role as a surface protein.

[0521] Due to the starch binding domain, Ortseifen (2016) (Ortseifen 2016) proposed that Cgt may be involved in the binding and retention of energy sources in the context of the Carbophore model (Wehmeier 2003). Evidence is also given here by RT-qPCR, which shows that the gene cgt is differentially expressed in glucose-, galactose- and lactose-grown cultures compared to cultures grown on maltose, higher maltodextrins and cellobiose. This is consistent with the differential proteome analysis on the carbon sources maltose and glucose (Wendler et al. 2015a; Wendler et al. 2015b). These results suggest a carbon-dependent expression of cgt. It will be exciting to elucidate the regulatory mechanisms. However, it remains to be considered that more than 900 genes are putatively involved in transcriptional regulation in A. aeruginosa SE50 / 110, of which 697 are annotated as transcriptional regulators (GenBank: LT827010.1) according to the annotation of Wolf et al. (2017b).

[0522] The sugar-dependent expression of cgt could indicate a function in the utilization of maltose, higher maltodextrins and - potentially - also cellobiose. However, our studies with the deletion mutant Δcgt did not reveal phenotypic differences regarding carbon utilization. This was tested for a total of 105 different carbon sources, 103 of which were analyzed in the OmniLog screening system and six in liquid culture.

[0523] Since the function of Cgt may be negligible under excess carbon source, but is essential when grown under conditions of limiting carbon source, the inventors have tested the growth of the deletion mutant ΔCgt and the wild type on a basic medium with low concentrations of starch. Starch was selected as a carbon source due to the starch binding activity of Cgt, which was confirmed in the starch binding assay herein. However, the growth phenotype of the mutant could not be observed under conditions of limiting carbon source.

[0524] Another function within sugar metabolism may be to bind insoluble crystalline substrates that may lead to structural changes, which increase the accessibility of the substrate and enhance the activity of other hydrolases such as amylases. This mechanism has been described in soil bacteria Serratia marcescens for chitin lysis (Vaaje-Kolstad et al. 2005) and in the brown thermophilic spore fungus Thermobifida fusca for cell decomposition (Moser et al. 2008). In the genome of the actinomycete SE50 / 110, several genes encode putative α-glycosidic functions, three of which, α-amylase / pullulanase AcbE, AcbZ and PulA, were shown to accumulate in the extracellular space (Wendler et al. 2015a). In addition, another small extracellular protein (ACSP50_6253) with unknown function and starch binding capacity was identified in a starch binding assay. By heterologous expression of extracellular amylase and enzyme assays in the presence and absence of both -Cgt and ACSP50_6253-, supporting functions during starch degradation can be examined in future experiments.

[0525] In addition to 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 through the Sec pathway as part of the leader peptide, and the second is considered to be required for multimerization. Although Cgt is unlikely to be physically anchored in the membrane (Wendler et al. 2015a), the Cgt protein may be retained as a multimer in the mycelial mesh due to reduced fluid flow. In this case, the starch binding domain may also serve as an anchor.

[0526] In the role of the putative surface protein, the inventors initially believed that it had a protective function under pH and osmotic stress or drought. However, screening experiments showed that in liquid culture, at different pH, the deletion of the cgt gene did not lead to significant growth inhibition. According to screening experiments on solid culture media, there was no evidence that Cgt might have a protective function under pH or drought.

[0527] Hints for putative function in the context of osmoregulation were given by reverse transcription quantitative PCR of wild-type grown on varying amounts of maltose. Here, the inventors observed that -1 In comparison, when -1 When grown on maltose, the transcription of gene cgt decreased by 2.9 times, which may be the effect of osmotic pressure. -1 to 681mOsmol kg -1 The growth of the deletion mutant Δcgt was analyzed in liquid cultures of 247 cells / mL. No differences in growth and viability were observed for the deletion mutant Δcgt compared to the wild type under all conditions tested.

[0528] Surprisingly, no matter when overdosing different carbon sources or under restrictive conditions, no matter under different pH or under osmotic pressure conditions, the deletion of cgt gene does not observe obvious physiological effects, and it is likely that the function of Cgt becomes obvious only in its natural environment and in the competition with other soil organisms. Interestingly, the inventors have found similar independent single CBM-20 domain proteins in 17 other prokaryotic species, most of which belong to Actinomycetales. Although rare, this at least shows a specific distribution, and shows that Cgt is not a strain-specific protein. Most species carrying single domain CBM-20 proteins are related to soil habitats. Together with the fact that cgt is highly expressed in actinomycetes SE50 / 110, this supports the hypothesis that proteins such as Cgt realize key functions in bacteria living in this habitat. The function of Cgt can be tested in the future by co-cultivation in direct contact with other microbial competitors.

[0529] Although surprisingly, Cgt proved to be unnecessary under the laboratory conditions tested, the inventors observed a positive phenotype for acarbose production. An increase in acarbose yield of 8.3% to 16.6% was obtained by deleting cgt. Although the final product yield varied slightly between batch cultures, the Δcgt mutant always performed significantly better. This was shown over a period of several months in three independent shake flasks and in several microcultures in maltose basal medium (data not shown). Thus, the improved production is robust over long periods of time and in different culture settings.

[0530] We propose that this is caused by the metabolic burden of cgt gene expression in the wild type, which brings relief of energy and free resources in Δcgt. These resources may be redirected to acarbose biosynthesis, which is a growth-related product. No direct regulatory effect of cgt deficiency on acb gene expression was observed.

[0531] Analysis of functional relevance of carotenoid formation

[0532] Light-dependent carotenoid formation and oxidative stress reduce acarbose production in Actinomycetes SE50 / 110 born

[0533] It is known that actinomycetes produce a variety of soluble pigments, including yellow, orange and pink pigments of the carotenoid class (Parenti and Coronelli 1979). The pigment of actinomycetes SE50 / 110 is orange. When exposed to light culture, its formation is enhanced. Since the pigment is also found in the supernatant, it seems to be soluble in aqueous solution. After cell extraction and separation by thin layer chromatography, spectral analysis showed that the absorption maximum was located at 450, 475 and 505-510, which was confirmed by the absorbance scan performed during HPLC separation. Consistent with these findings, computer simulation reconstruction showed that actinomycetes SE50 / 110 has a complete genetic apparatus for producing C40-carotenoids similar to sioxanthin from tropical salt spore CNB-440 (Richter et al., 2015, Wolf et al., 2017b) (Figure 21 and Table E12).

[0534] Table E12. Reconstruction of carotenoid synthesis in Actinoplanicum SE50 / 110. Two terpene synthesis gene clusters were identified by antiSMASH analysis (Blin et al. 2017; Weber et al. 2015), which can be attributed to the formation of C40-carotenoids similar to the sioxanthin gene cluster from tropical salina CNB-440 (Richter et al., 2015) (terpene clusters 1-2). In addition, the camphene-like monoterpene gene cluster (terpene cluster 3), all genes of the MEP / DOXP pathway and genes encoding lycopene degradation were identified by BlastP analysis (Altschul et al. 2005) and KEGG (Kanehisa et al. 2014).

[0535]

[0536]

[0537]

[0538] The genes for C40-carotenoid biosynthesis are organized into three gene clusters: terpene clusters 1, 2a, and 2b (see Fig.21D ).

[0539] In contrast to Salinosporium tropicalis, homologs of crtY and crtU encoding cyclases and desaturases could not be identified in A. pacificum SE50 / 110 (Wolf et al. 2017b). Instead, two cyclases of the CarR-domain superfamily were found in the present study. They are located in terpene cluster 2b (Figure 21). CarR-domain cyclases are common in fungal, archaeal and bacterial genomes (information obtained from CDD search of NCBI (Marchler-Bauer et al. 2017)). Since the pigment of SE50 / 110 is orange, the terminal cycle of the red precursor lycopene is highly likely and probably catalyzed by one or two CarR-domain cyclases. Similar to Salinosporium tropicalis, the carotenoid gene cluster of SE50 / 110 contains the glycosyltransferase CruC (Figure 21, Table E12). This strongly suggests that for the glycosylated carotenoids, the pigment appears to have a polar character based on the observations, as it was found in the supernatant ( Fig. 22 B).

[0540] Comparative genomic analysis performed by the software platform EDGAR 2.0 (Blom et al. 2016) showed a similar arrangement of terpene clusters in related species of the genus Actinomyces, while a different organization was found in Streptomyces (data not shown). Thus, the gene arrangement found in SE50 / 110 and CNB-440 (Richter et al. 2015; Wolf et al., 2017b) seems to be characteristic of the Micromonosporaceae family.

[0541] In addition, genes for the synthesis of the building blocks IPP and DMAPP via the MEP / DOXP-pathway (Table E12), genes encoding camphene-like monoterpene synthase (terpene cluster 3, Table E12) and 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 odorous substances (Yamada et al., 2015). The inventors observed that strong pigmentation was associated with production losses. This was confirmed by comparing the growth and acarbose yield of cultures exposed to light and protected from light ( Fig. 22 When inducing carotenoid formation, exposure to an intensity of 22-44 μE (1 μE = μmol 光子 m -2 s -1 ) light, the acarbose production and growth of A. acarbose SE50 / 110 was strongly reduced. Overall, a loss of 39% in the final acarbose concentration was monitored.

[0542] Deletion of merR in SE50 / 110 induces carotenoid formation without exposure to light

[0543] Since natural light or light can induce the formation of carotenoids ( Fig. 22 B, C), this study looked for possible regulatory genes in SE50 / 110. A MerR-regulator (ACSP50_0145, Fig.23 ).

[0544] The MerR-family consists mainly of activators that are able to respond to environmental stimuli such as oxidative stress, heavy metals or antibiotics (Brown et al., 2003). In fact, several members of the MerR-family have been described as light-dependent activators or repressors of carotenoid biosynthesis in non-photosynthetic bacteria, such as LitR in 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). Here, cobalamin (vitamin B12) acts as a cofactor, which mediates photosensitivity because it is able to absorb ultraviolet and blue light: by covalently binding to the regulator or shedding after light excitation, it is able to modulate the conformation and activity of the regulator (van der Horst et al., 2007). The regulatory mechanism and binding site are very different: while in Thermus thermophilus and Bacillus 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 released after light irradiation, LitR in Streptomyces coelicolor appears to be the main light-induced transcriptional activator of the adjacently located litS, which encodes the ECFσ factor and directs the transcription of carotenoid biosynthesis genes (Takano et al. 2005). The gene encoding the ECFσ factor is not present in the gene cluster of SE50 / 110. In the Gram-negative bacterium Myxococcus xanthus, the B12-dependent MerR regulon is part of a complex regulatory cascade that includes eight other regulatory genes (Fontes et al. 2003; Galbis-Martinez et al. 2012). In fact, no homologs of the regulatory network from Myxococcus xanthus were identified in the genome of SE50 / 110 by BLASTP-analysis (data not shown).

[0545] The MerR family regulator ACSP50_0145 of Actinomycetes SE50 / 110 contains an N-terminal HTH motif and a C-terminal B12 binding domain according to BLASTP analysis and CDD searches (Marchler-Bauer et al. 2015; Marchler-Bauer et al. 2010; Altschul et al. 2005). The location of the HTH domain constitutes a transcriptional repressor (Pérez-Rueda and Collado-Vides 2000).

[0546] CRISPR / Cas9 deletion of the corresponding gene in SE50 / 110 strongly induced carotenoid formation without exposure to light (Figure 24B, C). This confirms the function as a transcriptional repressor.

[0547] In fact, it must be noted that the repressor / operator system is leaky, since the typical orange color is produced in the wild type without exposure to light. Accordingly, under dark conditions, the transcription of the genes crtEBI and idi (ACSP50_0146-0149) is doubled only in ΔmerR compared to the wild type ( FIG. 24E ). These differences are significant for CrtE, CrtB and idi. No effect on the transcription of the acb gene was observed.

[0548] However, in the case of this study, the question of whether pigment formation in ΔmerR affects the formation of the fine chemical acarbose was examined. Again, higher carotenoid formation correlated with lower acarbose formation (Figure 24A, D). When illuminated, both wild type and ΔmerR were strongly colored, and the final acarbose concentration of both strains was similar, reaching approximately 0.52 g L -1 (Figure 24B, D). This corresponds to a 38% reduction in acarbose formation compared to the wild type under dark conditions (reaching 0.83 g L -1 ). This is consistent with previous growth experiments with the wild type described herein.

[0549] Under dark conditions, the acarbose produced by ΔmerR was higher than that of wild type (0.70 g·L -1 ) by about 15% (Figure 24D). This suggests that these production losses are due to the waste of resources for carotenoid formation in the deletion mutants (Figure 24C). In summary, the yield loss (38-39%) under light conditions may be caused by further light-induced stress in both the deletion mutants and the wild type.

[0550] Comparative transcriptome analysis of wild-type cultured in dark and light conditions using microarray technology revealed complex responses affecting the transcript levels of various genes (see Fig.25 ). Several differentially expressed genes indicate cellular responses against oxidative stress. Oxidative stress is caused by reactive oxygen species (ROS), which are formed by energy transfer (resulting in singlet oxygen) or electron transfer (resulting in 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).

[0551] In SE50 / 110, tyrosinase MelC (ACSP50_4950, formerly: ACPL_5017), a photoprotectant involved in the formation of the brown pigment eumelanin (Wolf et al. 2016), and genes for riboflavin biosynthesis (ACSP50_6437-40) were more strongly transcribed when exposed to light ( Fig.25 ). Riboflavin is a water-soluble photooxidative sensitizer 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 that are involved in cellular redox metabolism, light sensing, DNA repair and further functions (reviewed in Garcia-Angulo (2017)). Thus, riboflavin and its derivatives are important micronutrients that enable cells to overcome oxidative stress (Chen et al., 2013).

[0552] According to this, several flavin-dependent oxygenases were also transcribed more strongly when exposed to light. One of them was annotated as taurine dioxygenase, whose 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), which are able to capture ROS and act as redox buffers (Gout 2019). Correspondingly, other genes that may be involved in cysteine ​​and methionine metabolism and transport were transcribed more strongly in cells exposed to light.

[0553] Notably, several transcriptional regulator genes and the gene encoding the sigma factor SigE (ACSP50_0558) were also more strongly transcribed ( Fig.25 SigE has been implicated in oxidative stress responses in the photosynthetic bacterium Rhodococcus sphaeroides (reviewed in Ziegelhoffer and Donohue (2009)) and in envelope stress responses in the related species Streptomyces coelicolor (Hutchings et al. 2006) and Corynebacterium glutamicum (Park et al., 2008). SigE may be involved in oxidative stress responses in SE50 / 110.

[0554] Interestingly, compared with the wild-type of lucifuge, the gene of carotenoid biosynthesis and regulator MerR is not significantly stronger in the wild-type exposed to light. This is noteworthy because the obvious influence of light on carotenoid formation can be observed in the wild-type. Since carotenoid synthesis occurs under darkness and illumination, and the increase of relative transcript amount in the regulator mutant is quite moderate (see above), the influence on transcription level may not be obvious. It is inferred that there may be further regulation of carotenoid synthesis on protein level or metabolome level, such as degrading carotenoid or terpenoid precursor by carotenoid cleavage dioxygenase (ACSP50_5522). However, according to the result obtained from the microarray of wild-type, crt gene expression does not seem to be the main target of overall oxidative stress response, similar to the discovery from Rhodococcus spherical (reviewed in Ziegelhoffer and Donohue (2009)).

[0555] In conclusion, light exposure triggers the oxidative stress response and appears to have an important impact on the allocation of metabolic resources towards growth, carotenoid and acarbose formation. The regulation of carotenoid biosynthesis appears to be uncoupled from the overall response to oxidative stress, which requires further investigation. It is desirable to gain a better understanding of these processes in the future, considering the direction of metabolic flux towards the production of acarbose. The sigma factor SigE may be responsible for the oxidative stress response, as its transcription is higher when exposed to light.

[0556] In addition to light stress, this study confirmed that most of the production losses can be directly attributed to carotenoid formation. Carotenoids of non-photosynthetic bacteria are thought to function as photoprotectants (Lee and Schmidt-Dannert 2002), as they have shown protection against photodynamic killing (Mathews and Sistrom 1959). Since the influence of light can be excluded by simple structural measurements, carotenoid formation is considered unnecessary under laboratory conditions. To improve the production of acarbose, shutting down the co-occurring carotenoid biosynthetic pathway, for example by deleting the central gene crtI, can be used for strain development. Since carotenoids affect membrane fluidity (Gruszecki and Strzalka 2005), the lack of C40-carotenoids can also affect the surface and hyphal structure of A. spp. SE50 / 110. Regarding production, the disintegration of hyphal clumps is beneficial to increase the number of cells on the mycelial surface and biochemically available.

[0557] Overexpression of acbB and gtaB

[0558] The genes acbB and gtaB of the expression vector pSETT4 were tested. Both genes acbB and gtAB are likely involved in amino sugar synthesis, which is 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 thought to be involved in the supply of the precursor glucose-1P. Interestingly, both proteins showed increased protein amounts in the cytosol of acarbose producers.

[0559] pSETT4gap and pSETT4tip vectors for single gene overexpression

[0560] A new cloning system was implemented that allows easy cloning and overexpression of single genes in Actinomycetes strains such as Actinomycetes SE50 / 110. To this end, the strong promoter of the gene gapDH from Eggerthella tarda was cloned in front of the lacZ cassette in the pSET152-backbone. The gene lacZ is transcribed under the control of the lac-promoter and is flanked by recognition sites for the restriction enzyme BsaI, which enables the exchange of lacZ by the gene of interest by Gibson Assembly (Gibson et al. 2009), restriction / ligation cloning or Golden Gate cloning (Engler et al. 2008). Since strong expression requires strong termination, T4 terminators were introduced before and after the cloning site of the new expression system. The T4 terminator has been successfully used in the pGUS cloning system developed by Myronovskyi et al. (2011). The full track RNAseq analysis of pGUS integration mutants performed herein showed that the T4 terminator effectively blocks transcription and prevents read-through from the integrase gene into the gene of interest. As shown in preliminary experiments, the T4 terminator did not have any adverse effect on the transcription of the acb gene when introduced into A. planarians SE50 / 110 via pSET152 integration.

[0561] In addition, by sequencing the enriched primary transcript library derived from the promoter screening experiment, two putative promoters were identified behind the gene of interest in the antisense direction ( Fig.26 ). These two pseudo-promoters were removed in the new expression system to prevent antisense transcription. In addition, an additional (third) T4 terminator was introduced in the opposite direction behind the cloning site to prevent further putative antisense reading.

[0562] In order to allow the exchange of promoter sequences, NdeI and KpnI restriction sites were introduced. In this study, the strong gapDH-promoter was exchanged with the medium-strong TipA promoter from Streptomyces lividans. Thus, it was shown that the system can be easily modified, for example, adjusted for other species of the order Actinomycetales. The strong and medium-strong overexpression of the genes acbB and gtaB of the test vector (named after pSETT4gap and pSETT4tip) was performed.

[0563] Moderate overexpression of acbB causes improvement in acarbose formation

[0564] dTDP-D-glucose-4,6-dehydratase AcbB appears to be involved in the production of activated amino sugars from the feeder pathway of D-glucose-1P-acarbose biosynthesis ( Figure 1 ): It was found that increased AcbB-activity improves the supply of modified precursors: Briefly, two overexpression mutants were established based on the expression vector pSETT4 described elsewhere herein. In these mutants, acbB was transcribed under the control of the moderately strong tipA promoter or the strong gapDH promoter. As previously disclosed in (Schaffert et al., 2019), expression vectors using the native promoter did not lead to significant overexpression of genes of the ACB gene cluster. Therefore, the native promoter was used as a control in the pSET152- and pSET4-vector backgrounds. Growth and acarbose formation were monitored in two shake flask cultures in maltose basal medium ( Fig. 27 ).

[0565] Compared to the control strain, the mutant with acbB transcribed under the control of the heterologous tipA-promoter showed enhanced acarbose production: in two independent cultivations, the yield coefficient increased to 48.6% and 51.9% compared to the empty vector control ( Fig.28 By using the strong gapDH-promoter, the acarbose yield coefficient was slightly increased ( Fig.28 ).

[0566] 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 ( Fig.29 ). Therefore, the supply of activated glucose moieties seems to be guaranteed. In this mutant, the mass m / z = 545 [MH + ] increases ( Fig.29 , about 41%). Without being bound by theory, this intermediate accumulates by moderate AcbB overexpression, for example using pSETT4tip::acbB.

[0567] At the beginning of the growth phase, an increase in AcbB expression was observed within the expected range: the strongest overexpression was obtained using the gapDH-promoter (log2(fold change)=6.54) followed by the TipA promoter (log2(fold change)=4.06). Fig.30 The use of the native promoter did not result in a significant increase in the relative transcript amount of acbB. This was tested in the pSET152- and pSETT4-vector backgrounds ( Fig.30 Other genes in the acb gene cluster were not significantly affected, as shown by acbA and acbV ( Fig.30 The only exception was that the transcript abundance of acbA was slightly higher in pSETT4tip::acbB (log2(fold change) = 1.87).

[0568] It is noteworthy that the transcriptional profile in the linear growth phase differs from that in the early growth phase: here, only a doubling of the transcriptional amount was achieved by using the gapDH-promoter (log2(fold change)=2.05), whereas by using the tipA-promoter, overexpression of acbB was maintained, but to a lesser extent (log2(fold change)=3.33)( Fig.30 , Fig.31 ).

[0569] In overexpression mutants including heterologous promoters, the relative transcription of acbB decreased from 4.06-fold to 3.33-fold (log2 (fold change)) in pSETT4tip::acbB between two sampling times, and decreased from 6.54-fold to 2.05-fold in pSETT4gap::acbB. It is believed that although the transcription of the chromosomal AcbB copy is downregulated in these mutants, the transcription of the vector copy is maintained by the heterologous promoter. The difference in AcbB transcription at different sampling times further indicates that the downregulation of ACB gene transcription occurs more strongly and earlier in pSETT4gap::acbB compared to pSETT4tip::acbB, respectively. Overexpression of AcbB (pSETT4gap::acbB and pSETT4tip::acbB) appears to slow down during the linear growth phase.

[0570] In conclusion, especially in the culture medium, overexpression of acbB by using the tipA promoter seems to be beneficial for the production of acarbose, whereas strong overexpression by using the gapDH-promoter seems to have only a minor effect on the formation of acarbose. Further improvements in the formation of acarbose could be achieved by varying the expression level of acbB, for example by using alternative promoters from promoter screening or by introducing multiple gene copies.

[0571] In conclusion, this study demonstrates that moderate overexpression of AcbB increases acarbose yield, probably due to improved amino sugar supply. By moderate overexpression of acbB (e.g. by using the tipA-promoter), a positive effect on acarbose production was observed, producing about 50% more acarbose in two independent cultures. Thus, improvement of acarbose biosynthesis was achieved by overexpression of a single acb gene.

[0572] Moderate overexpression of gtaB leads to improved acarbose formation

[0573] GtaB is believed to catalyze the interconversion of UDP-glucose and glucose-1P. Surprisingly, it was found that overexpression of GtaB triggers acarbose formation. Without being bound by theory, this may occur by improving the distribution of the precursor glucose-1P. As shown in shake flask culture in maltose basal medium ( Fig.32 ), the final yield coefficient of acarbose of the gtaB overexpression mutant introduced into pSETT4tip increased to 8.56%.

[0574] Interestingly, acarbose formation increased specifically in the late linear growth phase to the stationary growth phase. In the overexpression mutant, the relative transcript amount of the gene gtaB increased 2.64-fold (log2(fold change)) ( Fig.33 ).

[0575] Since the metabolism of activated sugars is linked to or redirected to other metabolic pathways, they are not thought to accumulate. However, as shown in the previous experiments, the supply may be severely disturbed. Analysis of the intracellular metabolome showed similar amounts of phosphorylated hexoses and / or UDP-glucose ( Fig.34 ). Therefore, the activated C6-sugar pool is not significantly affected by gtaB overexpression.

[0576] Interestingly, the mass m / z = 545 [MH + ] was significantly reduced (by about 48%), which may correspond to dTDP-4-keto-6-deoxy-D-glucose, a proposed product of AcbB. This may indicate that the flow through the synthetic chain is more balanced, as the accumulation of this metabolite is reduced compared to the empty vector control and the AcbB overexpression mutant ( Fig.34 ). In conclusion, the introduction of a second gene copy of gtaB has a positive effect on acarbose production, but the effect of gtaB overexpression on the distribution of cellular substances remains unclear.

[0577] Transfer of this construct to a production strain of A. aeruginosa may lead to an increase in beneficial effects, since here the demand for the precursor is higher compared to the wild type. Since strong overexpression of AcbB leads to an imbalance in glucose-phosphate metabolism, it seems reasonable that combined overexpression of acbB and gtAB could further improve acarbose production beyond the effects observed with single overexpression.

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Claims

1. A method for engineering an Actinomyces strain for improving acarbose production, the method comprising engineering the Actinomyces strain to (i) making the expression of the extracellular small carbohydrate binding protein Cgt according to SEQ ID No. 20 absent or reduced; The Actinomycetes strain is an Actinomyces strain.

2. The method according to claim 1, comprising: (i) Deletion or mutation of the gene encoding the extracellular small carbohydrate binding protein Cgt according to SEQ ID No.

20.

3. An Actinomyces strain for producing acarbose, wherein the Actinomyces strain is an Actinomyces strain, and wherein the Actinomyces strain is genetically engineered so that the expression of the extracellular small carbohydrate binding protein Cgt according to SEQ ID No. 20 is absent or reduced.

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

5. Use of the Actinomycetales strain according to claim 3 in producing acarbose.

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