β-LACTAM PRODUCING STRAINS

Field of the invention

The present invention relates to β-lactam compound producing strains, to a method for their construction as well to the identification and inactivation of genes and enzymes, leading to an increased production efficiency of β-lactam compounds.

Background of the invention

Semi-synthetic β-lactam antibiotics (SSA's) are produced on an industrial scale starting from β-lactam compounds such as penicillinG (PenG), penicillinV (PenV), 6- aminopenicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7-ADCA), 7- aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem-4-carboxylate (7- ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3-cephem-4-carboxylate (7-PACA), 7- aminodeacetylcephalosporanic acid (7-ADAC), 7-amino-3-carbamoyloxymethyl-3- cephem-4-carboxylic acid (7-ACCCA) and others.

The production level of the β-lactam compounds in the commercially applied organisms has been increased considerably over the years. For instance, a modern Penicillium chrysogenum production strain is reported to produce about 40-50 g/l, whereas the original strains produced about 1 mg/l (Elander, R. P. (2002) University of Wisconsin contributions to the early development of penicillin and cephalosporin antibiotics SIM News 52, 270-278; Elander, R.P. (2003) Industrial production of β-lactam antibiotics. Appl Microbiol Biotechnol 61 , 385-392). This enhanced production level was realised by classical mutagenesis techniques (Elander, R. (1983) Strain improvement and preservation of β-lactam producing microorganisms in A. L. Demain and N. Solomon (eds.) Antibiotics containing the β-lactam structure I. Springer-Verlag, New York, N.Y., 97-146).

PenicillinG can be used as a starting point to make semi-synthetic penicillins (SSP's) as amoxicillin and ampicillin, but it can also be used to make semi-synthetic cephalopsorins (SSCs). The first generation 7-ADCA product was derived from PenG

whereby both the expansion of the 5-membered penem ring to the 6-membered cephem ring and the subsequent cleavage of the phenylacetic acid side chain of the phenylacetyl-7-ADCA were carried out using chemical reactions.

The next generation 7-ADCA product was still obtained from PenG but after the chemical ring expansion, the phenylacetic acid side chain of the phenylacetyl-7-ADCA was cleaved off enzymatically using a suitable (penicillin) acylase. Other processes have been developed wherein also the ring expansion of PenG to phenylacetyl-7-ADCA is carried out in vitro using a suitable expandase enzyme, but these processes are of little industrial importance. The most recent and most elegant production process for 7-ADCA comprises the culturing of a Penicillium chrysogenum, transformed with and expressing a gene encoding a suitable expandase. This engineered Penicillium chrysogenum strain, when grown in the presence of adipic acid as the side chain precursor in the fermentation vessel, produces and excretes adipyl-7-ADCA - see WO93/05158. In this production process, the adipyl-7-ADCA is recovered from the fermentation broth, subjected to a suitable acylase to cleave off the adipic acid side chain after which the 7- ADCA thus obtained is further purified, crystallized and dried. Other side chains precursors have been disclosed in WO95/04148 (2-(carboxyethylthio)acetic acid and 3- (carboxymethylthio)-propionic acid), WO95/04149 (2-(carboxyethylthio)propionic acid), WO96/38580 (phenyl acetic acid) and WO98/048034 and WO98/048035 (various dicarboxylic acids). The expandase takes care of the expansion of the 5-membered ring of the various N-acylated penicillanic acids, thereby yielding the corresponding N-acylated desacetoxycephalosporanic acids.

However, all these processes above rely on the efficiency of the host to convert the raw materials into β-lactams. And although more than 60 years of classical strain improvement have increased this efficiency enormously, the titers and yields are far away from other classical fermentation processes like lysine (with Corynebacterium glutamicum) and ethanol (with Saccharomyces cerevisae), resulting in a large percentage of the expensive raw materials (i.e. carbon, nitrogen, but also side chain precursors like adipate, phenylacetic acid, etceteras) being lost in the form of biomass or CO ₂ .

It has now surprisingly found that specific genes in a parent microbial strain capable of producing a β-lactam compound may be functionally inactivated which results in a more efficient production of the β-lactam compounds.

Detailed description of the invention

Group 1 is defined herein as the group with gDNA sequences with SEQ ID No 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 and 339. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 2 is defined herein as the group with gDNA sequences with SEQ ID No.2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 25, 29, 31, 34, 35, 37, 42, 43, 45, 47, 48, 49, 50, 57, 58, 59, 60, 61, 64, 65, 66, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,

137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,

154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,

171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,

188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 204, 205,

206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 247, 248, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 and 339. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 3 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 and 339. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 4 is defined herein as the group with gDNA sequences with SEQ ID No 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 29, 31, 33, 34, 35, 37, 42, 43, 45, 47, 48, 49, 50, 54, 57, 58, 59, 60, 61, 64, 65, 66, 68, 69, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 247, 248, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 and 339. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 5 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 67, 68, 69, 70, 71, 72, 73, 75, 76, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 137, 138, 140, 142, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 198, 199, 200, 202, 203, 204, 205, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 217, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 255, 256, 257, 258, 260, 263, 265, 266, 268, 269, 272, 273, 274, 275, 281, 282, 285, 286, 287, 288, 289, 290, 292, 293, 295, 296, 298, 304, 309, 310, 312, 313, 314, 315, 319, 322, 324, 325, 326, 327, 328, 329, 330, 336 and 337. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 6 is defined herein as the group with gDNA sequences with SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 264, 265, 266, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 313, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 and 339. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

Group 7 is defined herein as the group with gDNA sequences with SEQ ID No 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 16, 19, 20, 21, 22, 25, 29, 31, 34, 35, 37, 42, 43, 45, 47, 48, 49, 50, 57, 58, 59, 61, 68, 69, 71, 72, 73, 75, 76, 78, 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 133, 135, 137, 138, 140, 142, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 198, 199, 200, 202, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 219, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 247, 248, 251, 252, 253, 255, 256,

257, 258, 260, 265, 266, 268, 272, 273, 274, 275, 281 , 282, 285, 286, 287, 288, 289, 290, 292, 293, 295, 296, 298, 304, 309, 310, 315, 319, 322, 324, 325, 326, 327, 329, 330, 336 and 337. Table 1 summarizes these SEQ ID numbers together with their corresponding cDNA and protein sequences as well as their gene ID.

In a first aspect the invention provides a method for the identification of one or more genes of a parent microbial strain capable of producing one or more desired β- lactam compound(s) comprising the steps of a. Functionally inactivating the one or more genes in the parent microbial strain thereby generating one or more mutant microbial strains whereby each mutant microbial strain has at least one functionally inactivated gene; b. Culturing the parent microbial strain and the mutant microbial strains obtained in step (a) in a medium under conditions that allow production of the β-lactam compound; c. Measuring the concentration of the β-lactam compound and/or the carbon source

(e.g. sugar) and/or precursor in the culture medium of the parent microbial strain and the one or more mutant microbial strains; d. Selecting the mutant microbial strains which possess one or more of the desired properties selected from the group consisting of (i) an at least 10% higher concentration of the total β-lactam compounds in the culture medium compared to the parent microbial strain; and/or (ii) an at least 10% higher concentration of the one or more desired β-lactam compound(s) in the culture medium compared to the parent microbial strain; and/or (iii) an at least 10% improved yield (β-lactam / consumed sugar); and/or

(iv) an at least 5% improved yield in the one or more desired β-lactam compound(s) compared to undesired/total beta-lactam compound(s); and/or

(v) an at least 10% improved yield in the one or more desired β-lactam compound(s) on precursor (β-lactam / consumed precursor). e. Optionally identifying the gene(s) in the selected mutant microbial strain f. Optionally repeating step a. - d. whereby a selected mutant strain obtained in step d. is used as the parent strain in step a.

In the context of the present invention, the genes identified by the method of the first aspect of the invention are referred to as 'negative genes'.

"Functionally inactivating" or "functionally inactive" is defined herein as the inactivation of a gene which results in a residual activity of the encoded enzyme as compared to the activity in the parent strain of preferably less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 20%, more preferably less than 10%, more preferably less than 5%, more preferably less than 2%.

A "parent microbial strain" may be defined as a micro-organism capable of producing β-lactam compounds, preferably N-adipylated β-lactam compounds. A "mutant microbial strain" may be defined as a strain derived from the parent microbial strain by genetic engineering or classical mutagenesis.

The microbial strain capable of producing a β-lactam compound may be selected from the group consisting of a fungus, bacterium and yeast. Preferably the microbial strain of the present invention is a fungus, more preferably a filamentous fungus. A preferred filamentous fungus may be selected from the group consisting of Aspergillus, Acremonium, Trichoderma and Penicillium. More preferably the microbial strain of the present invention belongs to the species Penicillium, most preferably Penicillium chrysogenum. A preferred bacterium may be selected from the group consisting of Streptomyces, Nocardia, or Flavobacterium. In a preferred embodiment, the microbial strain of the present invention capable of producing an N-adipylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, which has been transformed with a gene encoding an expandase, preferably the Streptomyces clavuligerus cefE gene, which enables the strain to produce adipyl-7-ADCA when cultured in the presence of the precursor adipic acid. In another embodiment, the microbial strain of the present invention capable of producing an N-adipylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum and which, in addition to an expandase gene, preferably the Streptomyces clavuligerus cefE gene, has been transformed with a hydroxylase gene, preferably the Streptomyces clavuligerus cefF gene, whose expression product converts the 3-methyl side chain of adipyl-7-ADCA to 3-hydroxymethyl to give adipyl-7- aminodeacetylcephalosporanic acid (adipyl-7-ADAC). In another embodiment, the microbial strain of the present invention capable of producing an N-adipylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, has been transformed with a expandase/hydroxylase gene, preferably the

Acremonium chrysogenum cefEF gene, whose expression product converts the 3-methyl side chain of adipyl-7-ADCA to 3-hydroxymethyl, to give adipyl-7- aminodeacetylcephalosporanic acid (adipyl-7-ADAC). In another embodiment the microbial strain of the present invention capable of producing an N-adipylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, and in addition to the genes encoding expandase, preferably the Streptomyces clavuligerus cefE gene, and hydroxylase, preferably the Streptomyces clavuligerus cefF gene, is further transformed with an acetyltransferase gene, preferably the Streptomyces clavuligerus cefG gene, whose expression product (i.e. the acyltransferase) converts the 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain to give adipyl-7-ACA. In a further embodiment the mutant microbial strain of the present invention capable of producing an N-adipylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum and has been transformed with genes encoding an expandase, preferably the Streptomyces clavuligerus cefE gene, a hydroxylase, preferably the Streptomyces clavuligerus cefF gene and an O-carbamoyl transferase enzyme, preferably the Streptomyces clavuligerus cmcH gene, resulting in adipyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid.

The β-lactam compound produced by the microbial strain may be any β-lactam wherein the β-lactam moiety is a penem or cephem. Preferred embodiments of the one or more desired β-lactam compound(s) are those depicted in Figure 1. Most preferred β- lactam compounds are phenylacetyl- and adipyl-derivates of the intermediates listed before: 6-aminopenicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7- ADCA), 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem-4- carboxylate (7-ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3-cephem-4-carboxylate (7- PACA), 7-aminodeacetylcephalosporanic acid (7-ADAC), 7-amino-3- carbamoyloxymethyl-3-cephem-4-carboxylic acid (7-ACCCA) and others. Most preferred are N-adipylated cephalosporins, most preferred is adipyl-7-ADCA.

In step a) of the method of the invention, inactivation of the gene is defined as the modification of the gene in such a way so as to obtain a functionally inactive gene as defined hereinbefore. Methods for the modification of the gene in order to obtain the functionally inactive gene are known in the art and may include (but are not limited to): inactivation of the gene by base pair mutation resulting in a(n early) stop or frame shift; mutation of one or more codons which encode one or more a critical amino acids (such

as the catalytic triad for hydrolases); mutations in the gene resulting in mutations in the amino acid sequence of the enzyme which lead to a decreased half-life of the enzyme; modifying the mRNA molecule in such away that the mRNA half-life is decreased; insertion of a second sequence (i.e. a selection marker gene) disturbing the open reading frame; a partial or complete removal of the gene; removal/mutation of the promoter of the gene; replacing the promoter of the gene by a regulatable promoter; using anti-sense DNA or comparable RNA inhibition methods to lower the effective amount of mRNA in the cell. Most preferably the gene may be made functionally inactive by deletion resulting in a total absence of the encoded polypeptide and hence enzyme activity.

One approach is a temporary one using an anti-sense molecule or RNAi molecule (Kamath et al. 2003. Nature 421 :231-237). Another is using a regulatable promoter system, which can be switched off using external triggers like tetracycline (see Park and Morschhauser, 2005, Eukaryot Cell. 4:1328-1342). Yet another one is to apply a chemical inhibitor or a protein inhibitor or a physical inhibitor (see Tour et al. 2003. Nat Biotech 21 :1505-1508). The most preferred method is to remove part of or the complete gene(s) encoding the enzyme directly or indirectly mediating the incorporation efficiency of raw materials into the β-lactam compound. To obtain such a mutant one can apply state of the art methods like Single Cross-Over Recombination or Double Homologous Recombination. For this, one needs to construct an integrative cloning vector that may integrate at the predetermined target locus in the chromosome of the host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. The length that finally is best suitable in an experiment depends on the organism, the sequence and length of the target DNA., The efficiency of targeted integration of a nucleic acid construct into the genome of the host cell by homologous recombination, i.e. integration in a predetermined target locus, is preferably increased by

augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient hdfA or hdfB gene as described in WO 05/95624. WO 05/95624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration by preventing non-homologous random integration of DNA fragments into the genome. The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell. Alternative methods using second and/or lethal selectable markers are described in WO2008113847, WO2007115886 and WO20071 15887. Fungal cells may be transformed by protoplast formation, protoplast transformation, and regeneration of the cell wall. Suitable procedures for transformation of fungal host cells are described in EP 238023 and Yelton et at. (1984. Proc. Nat. Acad. Sci. USA 81 :1470-1474). Suitable procedures for transformation of filamentous fungal host cells using Agrobacterium tumefaciens are described by de Groot MJ. et al. (1998. Nat. Biotechnol. 16:839-842. Erratum in: Nat. Biotechnol. 1998. 16:1074). Other methods like electroporation, described for Neurospora crassa, may also be applied. Fungal cells are transfected using co- transformation, i.e. along with gene(s) of interest also a selectable marker gene is transformed. This can be either physically linked to the gene of interest (Ae. on a plasmid) or on a separate fragment. Following transfection transformants are screened for the presence of this selection marker gene and subsequently analyzed for the integration at the preferred predetermined genomic locus. A selectable marker is a product, which provides resistance against a biocide or virus, resistance to heavy metals, prototrophy to auxotrophs and the like. Useful selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC or sutB (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), as well as equivalents thereof. The most preferred situation is providing a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence (i.e. the selectionmarker gene) flanked at its 5' and 3' sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence. Cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment. To increase the relative frequency of selecting the

correct mutant microbial strain, a second DNA fragment comprising an expression cassette comprising a gene encoding a selection marker and regulatory sequences functional in the eukaryotic cell can be operably linked to the above described fragment (i.e. 5'-flank of target locus + selection marker gene + 3'-flank of target locus) and cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment and the absence of the second selection marker gene. The 5'- and 3'-flanks of the target locus can be for example the promoter and terminator of a gene, or the 5'- and 3'-end of the gene or any combination of these. The example provided in the present invention, as an illustration of the method, uses the promoter of the gene as 5'-flank and the gene as the 3'-flank to insert a selection marker between the promoter and gene, thereby disturbing (i.e. functionally inactivating) gene transcription. The gene sequences given above can be used to make similar functionally inactivated genes. The genes may be split in two, yielding a 5'-flank and a 3'-flank, but the gene may also be used to clone a larger piece of genomic DNA containing the promoter and terminator regions of the gene, which than can function as 5'-flank and a 3'-flank.

In step b) of the method of the invention, the parent and mutant microbial strains capable of producing a β-lactam compound may be cultured in a medium which allows the production of said β-lactam. These methods are well known to the person skilled in the art. One way of culturing said strains is described in the Examples. Depending on the β-lactam to be produced, the culture medium may comprise a side chain precursor, e.g. adipic acid or a suitable salt thereof for the production of adipylated β-lactam compounds (e.g. adipyl-6-APA, adipyl-7-ADCA, adipyl-7-ACA and others) or phenyl acetic acid for the production of penicillin G or other benzyl β-lactam compounds and so on. The culture medium furthermore may comprise a suitable carbon source such as a sugar (glucose and others).

In step c) of the method of the invention, the concentration of the β-lactam compounds may be measured during or after fermentation by methods well known to the person skilled in the art. In addition to the β-lactam compound, the microbial strains may also produce and excrete other so-called undesired β-lactam compounds. The sum of desired and undesired β-lactam compounds is referred to here as the total β-lactam compounds produced. Amongst the β-lactam compounds that may be measured are desired β-lactam compounds such as adipyl-7-ADCA and penicillinG and undesired β-

lactam intermediates or compounds such as L-α-aminoadipyl-L-cysteinyl-D-valine and isopenicillin as well as degradation products like 8-hydroxy-penillic-acid (see for an overview Figure 1 ). Other relevant concentrations that may be measured are the concentration of the carbon source such as sugar, the remaining concentration of the precursor as well as the concentration of degradation products of the precursor (for example ortho-hydroxyphenylacetic acid, OH-PAA). One way of measuring said concentrations is described in the Examples.

In step d) of the method of the invention, the mutant microbial strains capable of producing a β-lactam are selected for one or more of the desired properties cited. In one embodiment, mutant microbial strains are selected based on the concentration of the total β-lactam compounds produced in the culture medium under the conditions of the test. Preferably, the culture medium of the mutant microbial strain has an at least 10% higher concentration, more preferably an at least 20% higher concentration, more preferably an at least 30% higher concentration, more preferably an at least 40% higher concentration, more preferably an at least 50% higher concentration, more preferably an at least 100% higher concentration, more preferably an at least 200% higher concentration, more preferably an at least 300% higher concentration of the total β- lactam compounds compared to the concentration of the total β-lactam compounds produced by the parent microbial strain. In another embodiment, mutant microbial strains are selected based on the concentration of the one or more desired β-lactam compound(s) produced in the culture medium under the conditions of the test. Preferably, the culture medium of the mutant microbial strain has an at least 10% higher concentration, more preferably an at least 20% higher concentration, more preferably an at least 30% higher concentration, more preferably an at least 40% higher concentration, more preferably an at least 50% higher concentration, more preferably an at least 100% higher concentration, an at least 200% higher concentration, more preferably an at least 300% higher concentration of the one or more desired β-lactam compound(s) compared to the concentration of the one or more desired β-lactam compound(s) produced by the parent microbial strain. In a further embodiment, mutant microbial strains capable of producing a β- lactam are selected based on an at least 10% improved yield of total β-lactam on consumed sugar compared to the yield of the parent microbial strain (expressed as percentage - In Table 1 , this yield is referred to as Ybs). This yield is defined herein as ratio of the amount or millimoles of total β-lactam compounds found in the culture

medium and the amount or mole sugar consumed. The amount of total β-lactam compounds found in the culture medium may be calculated as follows:

(i) determining the molar concentration of each β-lactam compound in the culture medium and (ii) multiplying the value of i. by 1000;

(iii) sum up all the individual values for each β-lactam compound.

The amount of Cmol consumed sugar is calculated as follows:

(i) determining the concentration of sugar in the medium at the start of the cultivation; (ii) determining the concentration of sugar in the medium at the end of the cultivation;

(iii) subtracting the value of i. from the value of ii. (iv) dividing the value of iii. by the molecular weight of the sugar per Cmol (for glucose this is 30.03). In another embodiment, mutant microbial strains capable of producing a β-lactam are selected based on at least 5% improved yield in one or more desired β-lactam compound(s) with respect to the total β-lactam compounds produced compared to the yield of the parent microbial strain. For instance, when the parent microbial strain produces out of the 100% total β-lactam compounds only 80% of the desired β-lactam, the yield of the parent β-lactam producing strain is by definition 80%. The mutant microbial strain with an at least 5% improved yield produces the one or more desired β- lactam compound(s) with a yield of 84% (80 + 0.05 ^* 80). Preferably the improved yield is higher than at least 5%, preferably at least 10%, more preferably at least 20%. The maximal attainable improved yield depends on the yield of the parent microbial strain. In the example above, when the parent produces 80% desired β-lactam compound, the maximal improvement is 20% such that the mutant may produce 100% of desired β- lactam compound. In that case, the improvement of yield of the mutant β-lactam producing strain is 20/80=25%.

In a further embodiment, mutant microbial strains are selected based on an improved yield on precursor. It is well known in the art, that the precursors used for the production of the various β-lactam compounds (e.g. phenyl acetic acid, adipic acid etceteras), are not only incorporated in the desired β-lactam compounds, but may also be degraded via several metabolic routes for instance to serve as a carbon source to the microbial strain. Mutant microbial strains capable of producing a β-lactam are selected

based on an at least 10% improved yield of the desired β-lactam compounds on consumed precursor compared to the yield of the parent microbial strain. The yield is defined herein as ratio of the molar amount of the desired β-lactam compounds found in the culture medium and the molar amount of consumed precursor). The amount of millimoles desired β-lactam compounds found in the culture medium is calculated as follows:

(i) determining the concentration of each desired β-lactam compound in the culture medium in g/l;

(ii) dividing said concentrations by the specific molecular weight of each desired β- lactam compound;

(iii) multiplying the value of ii. by 1000;

(iv) sum up all the individual values for each desired β-lactam compound. The amount of mol consumed precursor is calculated as follows: (i) determining the concentration of precursor in the medium at the start of the cultivation;

(ii) determining the concentration of precursor in the medium at the end of the cultivation;

(iii) substracting the value of i. from the value of ii.;

(iv) dividing the value of iii. by the molecular weight of the precursor (for example, for adipate this is 146).

In step e) of the method of the invention, the gene that has been functionally inactivated in the selected mutant microbial strain may further be identified which may be necessary when the gene coding regions are not known at forehand. In a preferred embodiment however, part or the total of the genomic sequence of the parent β-lactam producing microbial strain may be determined. Analysis of the genomic sequences may then result in the determination of gene coding regions as well as the putative function of the enzymes encoded by the genes. The latter may be done by sequence comparisons with known gene- and/or protein sequences according to methods known in the art. In this embodiment it is possible to individually functionally inactivate one or more of at forehand selected, for instance based on the putative function, gene coding regions.

According to step f) of the method of the invention step a. - d. may be repeated one or several times whereby a selected mutant strain obtained in step d. of a previous round is used as the parent strain in step a. in a next round. Repeating steps a.-d. one or several times may increase the likelihood that the mutant β-lactam producing microbial

strain of the invention may contain more than one functionally inactivated genes and, as a consequence, may further be improved in one or more of the desired properties listed above.

In one embodiment, the method of the first aspect of the invention may also identify one or more genes of a parent microbial strain capable of producing a β-lactam compound that upon functionally inactivating the one or more genes in the parent microbial strain lead to a reduction and/or decrease of the desired properties defined hereinbefore, such as:

(i) an at least 10% lower concentration of the total β-lactam compound in the culture medium compared to the parent microbial strain; and/or

(ii) an at least 10% lower concentration of the desired β-lactam compound in the culture medium compared to the parent microbial strain; and/or (iii) an at least 10% decreased yield (β-lactam / consumed sugar); and/or (iv) an at least 5% decreased yield in desired β-lactam compound compared to undesired/total beta-lactam compound; and/or

(v) an at least 10% increased consumption of side-chain precursor. In the context of the present invention, these genes identified by the method of the first aspect of the invention are referred to as 'positive genes'.

In a second aspect, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound comprising the step of functionally inactivating one or more genes which may have been selected by the method of the first aspect of the invention and whereby the mutant microbial strain possesses one or more of the desired properties selected from the group consisting of i. an at least 10% higher concentration of the total β-lactam compound in the culture medium compared to the parent microbial strain; and/or ii. an at least 10% higher concentration of the one or more desired β-lactam compound(s) in the culture medium compared to the parent microbial strain; and/or iii. an at least 10% improved yield (β-lactam / consumed sugar); and/or iv. an at least 5% improved yield in desired beta-lactam compound compared to undesired/total beta-lactam compound; and/or v. an at least 10% improved yield in desired beta-lactam on precursor (β-lactam / consumed precursor).

The microbial strain capable of producing a β-lactam compound may be selected from the group consisting of a fungus, bacterium or yeast as has been described hereinbefore under the first aspect of the invention. Preferably, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound comprising the step of functionally inactivating preferably one or more genes selected from Group 1 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 1.

In a preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain gives an at least 10% higher concentration of the total β-lactam compound in the culture medium compared to the parent microbial strain, which method comprises the step of functionally inactivating preferably one or more genes selected from Group 2 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 2.

In another preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain gives an at least 10% higher concentration of the one or more desired β-lactam compound(s) in the culture medium compared to the parent microbial strain, which method comprises the step of functionally inactivating preferably one or more genes selected from Group 3 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 3.

In another preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has an at least 10% improved yield of total β-lactam on consumed sugar compared to the yield of the parent microbial strain which method comprises the step of functionally inactivating preferably one or more genes selected from Group 4 as defined hereinbefore or any gene which is at least 70%, more

preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 4. In another preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has an at least 5% improved yield in desired beta- lactam compound compared to undesired/total beta-lactam compound which method comprises the step of functionally inactivating preferably one or more genes selected from Group 5 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 5.

In a further preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain an has an at least 10% improved yield in desired β- lactam on precursor (β-lactam / consumed precursor) which method comprises the step of functionally inactivating preferably one or more genes selected from Group 6 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 6

In a highly preferred embodiment, the invention provides a method for the construction of a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has been improved in all 5 desired properties as defined hereinbefore and which method comprises the step of functionally inactivating preferably one or more genes selected from Group 7 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 7.

In another preferred embodiment of the method of the second aspect of the invention, the functionally inactivation of the 'negative genes' as defined hereinbefore, may be combined with an overexpression of one or more of the 'positive genes' as defined hereinbefore. This combination may lead to a further increase in one or more of the desired properties. Overexpression of the 'positive gene' is defined herein as the modification of the gene in such a way so as to obtain an activity of the enzyme by the positive gene as compared to the activity of the enzyme in the parent strain of preferably more than 100%, more preferably more than 110%, more preferably more than 120%,

more preferably more than 150%, more preferably more than 200%, more preferably more than 300%, more preferably more than 400%. Methods for the modification of the gene in order to obtain overexpression of the gene are known in the art and may include (but are not limited to): introduction of additional gene copies encoding host or heterologous proteins; over expression of host proteins from a strong promoter; modifying the transcriptional regulation of the genes controlling the genes involved in b- lactam production; mutation of critical amino acids leading to proteins with improved kinetic properties; mutations causing a increased half-life of the enzyme; modifying the mRNA molecule in such away that the mRNA half-life is increased; modifying the intracellular localization of the protein towards an organelle in which no products are present to inhibit its activity; introduction of one or more copies of heterologous genes encoding enzymes mediating b-lactam resistance. Preferably overexpression is obtained by introducing additional gene copies or driving gene transcription from a strong promoter.

In a third aspect, the invention provides a mutant microbial strain capable of producing a β-lactam compound characterized in that preferably one or more genes, preferably selected by the method of the first aspect of the invention, have been functionally inactivated, preferably by the method according to the second aspect of the invention and whereby the mutant microbial strain possesses one or more of the desired properties selected from the group consisting of i. an at least 10% higher concentration of the total β-lactam compound in the culture medium compared to the parent microbial strain; and/or ii. an at least 10% higher concentration of the one or more desired β-lactam compound(s) in the culture medium compared to the parent microbial strain; and/or iii. an at least 10% improved yield (β-lactam / consumed sugar); and/or iv. an at least 5% improved yield in desired beta-lactam compound compared to undesired/total beta-lactam compound; and/or v. an at least 10% improved yield in desired beta-lactam on precursor (β-lactam / consumed precursor).

Preferably, the invention provides a mutant microbial strain capable of producing a β-lactam compound characterized in that preferably one or more genes selected from Group 1 as defined hereinbefore or any gene which is at least 70%, more preferably at

least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 1 have been functionally inactivated.

In a preferred embodiment, the invention provides a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain gives an at least 10% higher concentration of the total β-lactam compound in the culture medium compared to the parent microbial strain characterized in that preferably one or more genes selected from Group 2 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 2 have been functionally inactivated.

In another preferred embodiment, the invention provides a mutant microbial strain capable of producing one or more desired β-lactam compound(s) and whereby the mutant microbial strain gives an at least 10% higher concentration of the one or more desired β-lactam compound(s) in the culture medium compared to the parent microbial strain characterized in that preferably one or more genes selected from Group 3 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 3 have been functionally inactivated.

In another preferred embodiment, the invention provides a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has an at least 10% improved yield of total β-lactam on consumed sugar compared to the yield of the parent microbial strain characterized in that preferably one or more genes selected from Group 4 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 4 have been functionally inactivated.

In another preferred embodiment, the invention provides a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has an at least 5% improved yield in desired beta-lactam compound compared to undesired/total beta-lactam compound characterized in that preferably one or more genes selected from Group 5 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least

95%, more preferably at least 99% homologous to the any of the gene sequences of Group 5 have been functionally inactivated.

In a further preferred embodiment, the invention provides a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain an has an at least 10% improved yield in desired β-lactam on precursor (β-lactam / consumed precursor) characterized in that preferably one or more genes selected from Group 6 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 6 have been functionally inactivated.

In a highly preferred embodiment, the invention provides a mutant microbial strain capable of producing a β-lactam compound and whereby the mutant microbial strain has been improved in all 5 desired properties as defined hereinbefore in step d(i) - d(v) of the method of the first aspect characterized in that preferably one or more genes selected from Group 7 as defined hereinbefore or any gene which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% homologous to the any of the gene sequences of Group 7 have been functionally inactivated.

In another preferred embodiment the mutant microbial strain of the third aspect of the invention as defined hereinbefore in addition to the one or more functionally inactivated genes (i.e. 'negative gene(s)' further comprises one or more 'positive gene(s)' as defined before which is over expressed as defined before.

It is defined herein that a sequence (sequence 1 ) is "substantially homologous" to another sequence (sequence 2) when sequence 1 possesses a degree of identity to sequence 2 of at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, still more preferably at least 96%, still more preferably at least 97%, still more preferably at least 98% and most preferably at least 99%. This definition of "substantially homologous" applies to nucleotide sequences as well as to amino acid sequences. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein are determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, for any DNA sequence determined by this automated approach, any nucleotide sequence

determined may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.

For the purpose of the present invention, the homology between two nucleotide sequences refers to the percentage of bases that are identical between the two sequences. DNA sequences related to the specified DNA sequences and obtained by degeneration of the genetic code are also part of the invention. Homologues may also encompass biologically active fragments of the full-length sequence.

The degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical between the two sequences. The degree of identity is determined using the BLAST algorithm, which is described in Altschul et al. (J. MoI. Biol. 215: 403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=- 4, and a comparison of both strands. A substantially homologous polypeptide may encompass polymorphisms that may exist in cells from different populations or within a population due to natural allelic or intra-strain variation. A substantially homologous polypeptide may further be derived from a fungus other than the fungus where the specified amino acid and/or DNA sequence originates from, or may be encoded by an artificially designed and synthesized DNA sequence. DNA sequences related to the specified DNA sequences and obtained by degeneration of the genetic code are also part of the invention. Homologues may also encompass biologically active fragments of

the full-length sequence. Substantially homologous polypeptides may contain only conservative substitutions of one or more amino acids of the specified amino acid sequences or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that can be altered in one of these sequences without substantially altering the biological function. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., (Science 247:1306-1310 (1990)) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein. The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cystein), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophane, histidine).

In a fourth aspect, the invention provides a process for the production of a β- lactam compound comprising culturing the mutant microbial strain of the third aspect of the invention in a fermentation medium under conditions suitable for the production of β- lactam compound . Such conditions are well known to the skilled person.

Figure legends

Figure 1 is an overview of the various enzymes and intermediates in β-lactam biosynthesis routes (i.e. penicillins, cephalosporins and cephamycins). The arrows represent the following enzymes: 1 , L-α-aminoadipyl-L-cysteinyl-D-valine synthetase; 2, isopenicillin N synthase; 3, acyl-CoA:6-aminopenicillanic acid/isopenicillin N acyltransferase; 4, PenN epimerase; 5, deacetoxycephalosporin C synthase (expandase); 6, deacetylcephalosporin C hydroxylase; 7, 3'-hydroxymethylcephem-O- carbamoyltransferase; 8, O-carbamoyl-deacetylcephalosporin C hydroxylase; 9, methyltransferase; 10, acetyltransferase; 1 1 , phenylacetyl-CoA ligase; 12, adipyl-CoA- ligase. The capitals in brackets represent the following intermediates: [A], L-α- aminoadipic acid; [B], L-cysteine; [C], L-valine; [D], L-α-aminoadipyl-L-cysteinyl-L-valine; [E], iso-penicillinN; [F], Penicillin G; [G], Penicillin N; [H], deacetoxycephalosporin C (DAOC); [I], deacetylcephalosporin C (DAC); [J], O-carbamoyl-DAC; [K], 7-α-hyroxy- OCDAC; [L], cephamycin C; [M], cephalosporin C; [N], adipyl-6-aminopenicillinic acid (Ad-6-APA); [O], adipyl-7-aminodeacetoxycephalosporanic acid (Ad-7-ADCA); [P], adipyl^-aminohydroxycephalosporanic acid (Ad-7-AHCA); [Q], adipyl-7-amino-3- carbomoyloxymethyl-3-cephem-4-carboozylic acid (Ad-7-ACCA); [R], phenylacetic acid; [S]. phenylacetyl-CoA; [T], adipic acid; [U], adipyl-CoA.

Figure 2 is a representation of the steps involved in deleting the Penicillium chrysogenum gene Pc12g00100. Legend: solid arrow, Pc12g00100 promoter; open arrow, Pc12g00100 ORF; hatched box, trpC terminator; dashed box, ccdA gene; solid box, lox site; crosses, recombination event; downwards arrows, subsequent steps in the procedure; REKR and KRAM, overlapping non-functional amdS selection marker fragments; REKRAM, functional amdS selection marker gene. Numbers indicate the SEQ ID No.'s of the oligonucleotides, "tag" indicates the presence of a specific nucleotide sequence which can be used for mutant identification.

Figure 3 is a representation of the steps involved in confirming the actual deletion of the Penicillium chrysogenum gene Pc12g12120. Legend: solid arrow, Pc12g12120 promoter; open arrow, Pc12g12120 ORF; hatched box, trpC terminator; solid box, lox site; REKRAM, functional amdS selection marker gene. Numbers indicate the SEQ ID NO.'s of the oligonucleotides for the three PCR reactions indicated (see also table 2).

Materials and Methods

Standard DNA procedures were carried out as described elsewhere (Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2 ^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) unless otherwise stated. DNA was amplified using the proofreading enzyme Physion polymerase (Finnzymes). Restriction enzymes were from Invitrogen or New England Biolabs. Fungal growth was performed in a mineral medium, containing (g/L): glucose (5); lactose (35); urea (4.5); (NH ₄ ) ₂ SO ₄ (1.1 ); Na ₂ SO ₄ (2.9); KH ₂ PO ₄ (5.2); K ₂ HPO ₄ (4.8) and 10 mL/L of a trace element solution containing (in g/l): citric acid (150); FeSO ₄ .7H ₂ O (15); MgSO ₄ .7H ₂ O (150); H ₃ BO ₃ (0.0075); CuSO ₄ .5H ₂ O (0.24); CoSO ₄ JH ₂ O (0.375); ZnSO ₄ .7H ₂ O (5); MnSO ₄ -H ₂ O (2.28); CaCI ₂ .2H ₂ O (0.99); pH before sterilization 6.5.

EXAMPLES

Example 1 Deletion of Penicillium chrysogenum gene Pc12g00100 (SEQ ID No 11)

In order to prevent the transcription of gene Pc12g00100 (SEQ ID No 1 1 ) a selection marker gene was inserted between the promoter and the open reading frame (ORF). To this end the promoter and the ORF were PCR amplified using the oligonucleotides SEQ ID No. 1028 plus 1367 and SEQ ID No. 1706 plus 2045, respectively (see Fig. 2 and Table 1 ). Phusion Hot-Start Polymerase (Finnzymes) was used to amplify the fragments. The fragments obtained are 1539 and 2515 basepairs (bp) in length (SEQ ID No 2374 and SEQ ID No 2375) and contain a 14 bp tail suitable for the so-called STABY cloning method (Eurogentec). From the standard STABY vector, pSTC1.3, two derivatives were obtained. One, pSTamdSL (SEQ ID No 2378), was used for cloning the PCR amplified promoter (SEQ ID No 2374). The other, pSTamdSR (SEQ ID No 2379), was used for cloning the PCR amplified ORF (SEQ ID No 2375). pSTamdSL was constructed by insertion of an inactive part of the amdS selectionmarker gene (see for example the PgpdA-amdS cassette of pHELY-A1 in WO04106347) by PCR amplification of the last 2/3 of the gene {amdS) and cloning it in the H/nc/lll-SamHI sites of pSTC1.3. pSTamdSR was constructed by insertion of another inactive part of the amdS selectionmarker gene (see for example the PgpdA-amdS cassette of pHELY-A1 in WO 04106347): the PgpdA promoter and the first 2/3 of the gene wherein the EcoRV sites where removed and cloning it in the H/nc/lll-Pmel sites of pSTC1.3. Also, a strong terminator was inserted in front of the PgpdA-amdS; the trpC

terminator was PCR amplified and introduced via the Sbf\-Not\ sites of the PgpdA-amdS fragment. Both vectors do contain an overlapping but non-functional fragment of the fungal selectionmarker gene amdS, encoding acetamidase and allowing recipient cells that recombine the two fragments into a functional selectionmarker to grow on agar media with acetamide as the sole nitrogen source (EP 635,574; WO97/06261 ; Tilburn et al., 1983, Gene 26: 205-221 ). The promoter and ORF PCR fragments (SEQ ID No 2374 and SEQ ID No 2375) were ligated into the respective vectors (pSTamdSL and pSTamdSR, repectively) overnight using T4 ligase (Invitrogen) at 16 ⁰ C, according to the STABY-protocol (Eurogentec) and transformed to chemically competent CYS21 cells (Eurogentec). Ampicillin resistant clones were isolated and used to PCR amplify the cloned fragments fused to the non-functional amdS fragments (see Fig. 2). This was done using the oligonucleotides SEQ ID No 2380 and SEQ ID No 2381. The thus obtained PCR fragments (SEQ ID No 2376 and 2377) were combined and used to transform a derivative Penicillium chrysogenum strain DS17690 (S917), deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands on April 15, 2008 with deposition number CBS 122850, with the hdfA gene deleted (according to the method described in WO05095624). In this strain the non-homologous end-joining pathway is disturbed and therefore the random integration of DNA is drastically reduced. And as the combined PCR fragments themselves should recombine also to form a functional amdS selection marker gene (i.e. the so-called bipartite or split-marker method), correct targeted integrants should undergo a triple homologous recombination event (see Fig. 2). Transformation was performed in microtiterplates as described in WO2008000715) and one transformant was subsequently transferred to a second acetamide selection plate (without KCI). After sporulation this selected mutant was replicated to a third acetamide selection plate (without KCI). For storage at -20 or -80 degrees Celsius the mutant colonies on these plates were covered with 10% glycerol.,

The same methodology was followed for all genes in table 1 and transformants were obtained for all of them.

Table 1.

Overview of genes identified in Penicillium chrysogenum by the method of the first aspect of the invention. The first column gives the unique Gene ID of each identified gene. Columns 2-7 summarize the results (expressed as percentage relative to the control = parent strain) obtained upon functionally inactivating the corresponding gene.

Column 2 gives the relative concentration of the total β-lactam compounds, column 3 gives the relative concentration of the desired β-lactam compound (adipyl-6-APA), column 4 gives the yield on sugar as defined in the text, columns 5 and 6 give the yield of the desired β-lactam compound (adipyl-6-APA) relative to the total β-lactam compound (column 5) and relative to the control = parent strain (column 6). Column 7 gives the yield of β-lactam on precursor as defined in the text. Cells in columns 2-7 which are highlighted (grey background and black font) contain values for each property which fulfil the criteria listed in step d(i) - d(v) of the method of the first aspect of the invention. Columns 8-14 summarize the SEQ ID Numbers for each identified gene. Column 8 gives the SEQ ID No for the genomic DNA sequence of each gene, column 9 the SEQ ID No for the corresponding Coding sequence (i.e. without the putative introns) and column 10 the SEQ ID No for the corresponding protein sequence. Columns 1 1-14 give the SEQ ID No for the oligonucleotides used as primers for each identified gene to obtain the fragments for functionally inactivating the respective gene as described in detail in example 1.

Example 2

Deletion of specific Penicillium chrysogenum genes leading to an increase in one or more desired properties

The transformants obtained in example 1 where inoculated in liquid media with 0.5% of adipate as the side chain precursor for the desired β-lactams and grown for 168 hours at 25°C. Subsequently, the cells were removed by centrifugation and 1 ml of the supernatant was used for NMR analysis. Quantitative ¹ H NMR experiments were performed at 600 MHz on a Bruker Avance 600 spectrometer. To a known quantity of filtrate, a known quantity of internal standard (for example maleic acid), dissolved in phosphate buffer was added prior to lyophilisation. The residue was dissolved in D ₂ O and measured at 300 ⁰ K. The delay between scans (30 s) was more than 5 times T ₁ of all compounds, so the ratio between the integrals of the compounds of interest and the integral of the internal standard is an exact measure for the quantity of the penicillins, intermediates (6-APA and IsopenicillinN), degradation products (8-HPA), remaining sugar and remaining side-chain (adipate).

As a control the parent strain P. chrysogenum δhdfA was cultivated and processed in the same way. All relevant results for the desired properties (total β-lactam, ad-6-APA, Ybs (yield of β-lactam on sugar), ratio desired vs. total β-lactam and yield of β-lactam on precursor) are displayed in table 1 , relative to the value of the control strain. All mutants are improved in 1 , 2, 3, 4 or all 5 desired properties as compared to the parent strain.

Example 3

Correct deletion of Penicillium chrysogenum gene Pc12g12120 leads to an increase in β-lactam production

The mutants obtained in example 2 were in colony purified and used for further characterisation: verification of the actual gene deletion by PCR and confirmation of the increased β-lactam titer after cultivation in 25 ml batch cultures (in 100ml shake flasks).

To isolate chromosomal DNA of the correct quality (i.e. enabling PCR amplification up to 9 kb) spores of mutant Pc12g12120 were used to inoculate 3 ml of medium in a 24-well MTP plate and grown for 2-3 days at 550 rpm, 25°C and 80% humidity. Cells are washed and protoplasted using standard buffers (see Swinkels, B.W., Selten, G. C. M., Bakhuis, J. G., Bovenberg, R.A.L., Vollebregt, A.W. 1997. The use of homologous amdS genes as selectable markers. WO9706261 ). Protoplastation was done for 2 hours at 37°C, using Glucanex at 10 mg/ml in the 24-well plates. Cells (protoplasts and remaining mycelium) are washed again and DNA was isolated using the Puragen DNA isolation kit (Gentra) according to the suppliers' instructions. The DNA was air-dried and dissolved in 100 ul water. PCR reactions were performed in a final 50 μl, with the following composition:

5 μl DNA template (5x diluted from sample preparation) 21.5 μl water

10 μl GC buffer (Finnzymes)

1 μl dNTP (stocksolution of 10 mM)

2 μl DMSO

5 μl Forward oligonucleotide (stock solution of 2 μM) 5 μl Reverse oligonucleotide (stock solution of 2 μM)

0.5 μl Phusion ^® Polymerase (Finnzymes)

To verify the correct deletion 3 PCR reactions are performed (see Figure 3). The first PCR reaction is to confirm the correct integration at left flanking, using for the locus of gene Pc12g12120 the specific forward oligonucleotide of SEQ ID NO 2382 and the reverse oligonucleotide of SEQ ID NO 2383; the former being specific for this gene locus and choosen just upfront of the fragment used for gene targeting and the latter annealing in the amdS selection marker, which can be used to verify all individual gene mutations. The second PCR reaction is to confirm the correct integration at right flanking, using for the locus of gene Pc12g12120 the specific reverse oligonucleotide of SEQ ID NO 2385 and the forward oligonucleotide of SEQ ID NO 2384; the former being specific for this gene locus and choosen just downstream of the fragment used for gene targeting and

the latter annealing in the amdS selection marker, which can be used to verify all individual gene mutations. The third PCR reaction is to confirm the absence of the WT fragment and the correct integration at the locus of gene; for this one can combine the two locus specific oligonucleotides of the first two PCR reactions, i.e. in the case of locus of gene Pc12g12120 the forward oligonucleotide of SEQ ID NO 2382 and the reverse oligonucleotide of SEQ ID NO 2383; if the gene targeting is correct this yields a much larger band than in the case of the WT (see table 2).

The PCR amplification is performed in a Tetrad machine of Biorad using the following program:

Step 1 : 30 sec at 98°C Step 2: 10 sec at 98°C Step 3: 30 sec at 55°C Step 4: 1.5-4.5 min at 72°C

(the actual extention time is set by using 0.5 min/kb to be amplified) Step 5: Repeat steps 2-4 for 35 cycles Step 6: 10min at 72°C

Table 2. Expected and observed PCR fragments in mutant at gene locus Pc12g12120 (see also Figure 3).

The results depicted in table 2 clearly demonstrate that mutant Pc12g12120 is correctly targeted and no WT gene is present. The same procedure can be applied to all the mutants identified and obtained according to the present invention; in all cases the oligonucleotides of SEQ ID NO 2383 and 2384 stay the same, but the oligonucleotides of SEQ ID NO 2382 and SEQ ID NO 2385 are gene specific.

The spores of thus proven correct mutant of locus Pc12g12120 were inoculated in 25 ml medium as described in example 2, in 100 ml shake flasks and incubated for 168 hours at 25 ⁰ C. and 280 rpm. As a control strain DS17690 was inoculated and grown in the same way. Subsequently, the cells were removed by centrifugation and 1 ml of the supernatant was used for NMR analysis. Quantitative ¹ H NMR experiments were performed at 600 MHz on a Bruker Avance 600 spectrometer. To a known quantity

of filtrate, a known quantity of internal standard (for example maleic acid), dissolved in phosphate buffer was added prior to lyophilisation. The residue was dissolved in D ₂ O and measured at 300 ⁰ K. The delay between scans (30 s) was more than 5 times T ₁ of all compounds, so the ratio between the integrals of the compounds of interest and the integral of the internal standard is an exact measure for the quantity of the penicillins, intermediates (6-APA and IsopenicillinN), degradation products (8-HPA), remaining sugar and remaining side-chain (adipate). Compared to the DS17690 strain, the mutation of gene Pc12g12120 improved the adipoyl-6APA titer significantly. With the titer of DS17690 set at 100, a duplo experiment of the Pc12g12120 mutant resulted in 125% and 128% adipoyl-6APA. Therefore, we may conclude that this mutant is improved in 1 , 2, 3, 4 or all 5 desired properties as compared to the parent strain and thus gene Pc12g12120 qualifies as a negative gene.

I Applicant's or agent's file reference number 26712-WO-PCT International application No.

EVDICATIONS RELATING TO A DEPOSITED MICROORGANISM

A. The indications made below relate to the microorganism referred to in the description first mentioned on page 26 line 16.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

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CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country) Uppsalalaan 8 P.O. Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 15/04/2008 Accession Number CBS 122850

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We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed to be withdrawn.

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Authoπzed officer

De Bie, Nicole

Form PCT/RO/134 (July 1992)