Genetically modified lignocellulolytic clostridium acetobutylicum

Abstract

The present invention relates to a genetically modified Clostridium acetobutylicum capable of growing on lignocellulosic biomass wherein a cip-cel operon is overexpressed, the expression level of each gene of said cip-cel operon in the genetically modified Clostridium acetobutylicum being higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum, the invention also relates to the enhancement of the growth of its derivatives on lignocellulosic biomass by further modification, and the use of these strains to produce bulk chemicals (e.g., ethanol, butanol, glycerol, 1, 2-propanediol, acetone, isopropanol, isobutylene, hydrogen, acetic acid, lactic acid, etc.) by conversion of lignocellulose as a carbon source.

Description

Technical Field

[0001] The present invention relates to the field of bioproduction. The present invention relates to a genetically modified strain Clostridium acetobutylicum, which is able to grow on lignocellulosic biomass because the expression level of the cip-cel operon of the strain is higher than the expression level in the corresponding non-genetically modified Clostridium acetobutylicum. The present invention also relates to the use of the genetically modified strain Clostridium acetobutylicum to produce bulk chemicals (such as ethanol, butanol, glycerol, 1,2-propylene glycol, 1,3-propylene glycol, acetone, isopropanol, hydrogen, acetic acid and lactic acid, etc.) by converting lignocellulose as a carbon source. Background Art

[0002] For nearly 100 years, Clostridium acetobutylicum (C. acetobutylicum) has been known to produce solvents such as butanol and other commodity chemicals. However, although C. acetobutylicum can utilize all sugars produced by the hydrolysis of cellulose (Compere and Griffith 1979) and hemicellulose (Dunning and Lathrop 1945; Sticheblykina and Nakhamanovich, BM 1959) and degrade polymers such as starch or xylan, it is unable to hydrolyze and grow on crystalline cellulose or pretreated lignocellulosic biomass (Lee, Forsberg and Gibbins 1985).

[0003] The cellulosome is a macromolecular complex for cellulose degradation (Bayer EA, Y. Shoham and R. Lamed. 2000) that has been genetically and biochemically characterized in several Clostridia, including Ruminiclostridium cellulolyticum (Gal, L&al. 1997), Clostridium cellulovorans (Doi, RH&al. 1994), Clostridium josui (Kakiuchi, M.&al. 1998) and Clostridium thermocellum (Lamed, R., and EA Bayer. 1988).

[0004] Surprisingly, Clostridium acetobutylicum ATCC 824 ( et al., 2001) revealed the presence of a large cellulosomal gene cluster. Sequence analysis showed that the cluster contained the following genes: the cipA scaffold protein gene, the Cel48A cellulase gene, several family 5 and 9 cellulase genes, the Man5G mannanase gene, and the hydrophobin OrfXp gene. The genetic organization of this large cluster is very similar to that of Archaea cellulolytica and Clostridium cellulophagus ( JP & al. 1997; Tamaru, Y. & al. 2000).

[0005] Since C. acetobutylicum cannot grow on cellulosic substrates, the presence of a cellulosomal gene cluster in the genome raises questions about its expression, function, and evolution. In earlier studies, we have determined that C. acetobutylicum can produce cellulosomal genes with an apparent molecular weight of approximately 665 kDa (Sabathé, F. & al. 2002). Biochemical and immunochemical analysis of cellulosomal components indicated the presence of at least four subunits, including the CipA scaffold protein and three major cellulases, Cel48A, Cel9X, and Cel9C cellulases.

[0006] According to EP2436698A1, the cellulases Cel9X and Cel9C of C. acetobutylicum are active on crystalline cellulose, while Cel48A of C. acetobutylicum is inactive, which explains why C. acetobutylicum cannot grow on crystalline cellulose or pretreated lignocellulosic biomass. To solve this problem, EP2436698A1 provides a genetically modified C. acetobutylicum containing a cel48SAFA hybrid gene (composed of a cel48A gene, wherein the sequence encoding the catalytic domain is replaced by the catalytic domain of the cel48F gene).

[0007] However, even when the native promoter of the cip-cel operon was replaced with a strong promoter, the expression level of the cel48SAFA hybrid gene was still relatively low and the ability of C. acetobutylicum to grow on cellulose was limited.

[0008] Tao et al. (TAO XUANYU et al.: "Precise promoter integration improvescellulose bioconversion and thermotolerance in Clostridium cellulolyticum", METABOLIC ENGINEERING, Vol. 60, July 1, 2020 (2020-07-01), pp. 110-118, ISSN: 1096-7176, DOI: 10.1016 / j.ymben.2020.03.013) disclosed an engineered R. cellulolyticum (abbreviated as "R. cellulolyticum", which is the new name for Clostridium cellulolyticum, as demonstrated by Yutin N et al., "A genomic update on clostridial phylogeny: Gram-negative sporeformers and other misplaced clostridia". Genomics update. Volume 15, Issue 10, October 2013, Pages 2631–2641, https: / / doi.org / 10.1111 / 1462-2920.12173), in which a synthetic P4 promoter or a predicted P2 promoter was inserted between the orfX and cel9H genes of the cip-cel gene cluster, resulting in increased expression of downstream genes of the cip-cel gene cluster, i.e., increased expression of genes cel9J, cel9M, and cel5N, but no increase in expression of upstream genes (e.g., cel48F). This resulted in enhanced cellulolytic activity.

[0009] However, in contrast to A. cellulolyticus, C. acetobutylicum and other related saccharolytic clostridia are able to utilize sugars at a very high metabolic rate ( Desvaux, Clostridium cellulolyticum: model organism of mesophilic cellulolyticclostridia, FEMS Microbiology Reviews, Volume 29, Issue 4, September 2005, Pages 741-764, https: / / doi.org / 10.1016 / j.femsre.2004.11.003), which makes them more suitable candidates as comprehensive biological treatment (CBP) organisms.

[0010] However, Archaea cellulolytica (the model species used by Tao et al.) and Clostridium acetobutylicum belong to two different major evolutionary groups of cellulosomal mesophilic bacteria, the first group including Archaea cellulolytica (the species used by Tao et al.), Clostridium sp. BNL1100, C. papyrosolvens and C. aquatica, while the second group includes C. acetobutylicum, C. cellulophagus, C. bornimense and C. saccharoperbutylacetonicum. In the second group, only C. cellulophagus is able to utilize cellulose. The two major different evolutionary groups of cellulosomal mesophilic bacteria differ in the structure of the cip-cel operon, its major scaffold protein genes and the regulation of the cip-cel operon by different promoters (see Dassa, Bareket, Ilya Borovok, Vincent Lombard, Bernard Henrissat, Raphael Lamed, Edward A. Bayer and Sarah 2017. "Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme" Microorganisms 5, no. 4: 74. https: / / doi.org / 10.3390 / microorganisms5040074).

[0011] Given this evolutionary and structural difference, one would not have expected that the same approach would be used to enhance cellulolytic activity in C. acetobutylicum.

[0012] More importantly, Sabathé, F. et al., 2002 described that the cellulosomes of C. acetobutylicum are inactive, and EP2436698A1 discloses that Cel48A of C. acetobutylicum is inactive. Increasing the expression of a complex that has been shown to be inactive, of which Cel48A is the main component, obviously cannot be expected to produce cellulolytic activity, since Tao et al. only showed a slight increase in the growth rate of bacteria on cellulose that were already able to grow on cellulose.

[0013] Finally, Mingardon et al. (Mingardon F. et al. "The Issue of Secretion in Heterologous Expression of Clostridium cellulolyticum Cellulase-Encoding Genes in Clostridium acetobutylicum ATCC 824". APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2011, pp. 2831-2838, doi: 10.1128 / AEM.03012-10) showed that overexpression of the cel48A gene in Clostridium acetobutylicum was toxic to the bacteria, further indicating that using a method similar to that disclosed by Tao et al. in Clostridium acetobutylicum would not be successful.

[0014] Therefore, there remains a need for improved C. acetobutylicum strains that can efficiently grow on crystalline cellulose or pretreated lignocellulosic biomass. Summary of the invention

[0015] In the context of the present invention, the inventors surprisingly found that, contrary to the teaching of EP2436698A1, the cel48A gene of Clostridium acetobutylicum encodes a functional Cel48A protein that is actually active on crystalline cellulose. In addition, the inventors found that the overexpression of the entire cip-cel operon (with natural genes, but replacing its common promoter with the promoter of the thlA gene as a strong constitutive promoter, such as in the strain SC1 disclosed in Example 1) or at least the overexpression of cipA, cel48A and orfxp (such as in the strain SC2 disclosed in Example 1) leads to the production of active cellulosomes, and the resulting strain is able to grow on lignocellulosic biomass. They also found that when under the control of the strong constitutive promoter of the thlA gene, the production level of the natural Cel48A protein is significantly higher than the hybrid Cel48SAFA protein. The ability to grow on lignocellulosic biomass can be further improved by overexpressing the cel9X gene and overexpressing the cel5Y gene and / or the xynB gene, and / or deleting the extracellular protease encoding gene nrpE. By increasing the production of CipA (such as in strain SC10, including an additional copy of the cipA gene controlled by the thlA gene promoter, as disclosed in Example 10), this allows for higher amounts of cellulosomes and lower amounts of free cellulases, thereby increasing the ratio of CipA scaffold proteins to cellulases in the cellulosomes, and further improving the ability to grow on lignocellulosic biomass. This also allows for efficient growth on crystalline cellulose.

[0016] In a first aspect, the present invention relates to a genetically modified Clostridium acetobutylicum capable of growing on lignocellulosic biomass, wherein the cip-cel operon is overexpressed, and each gene of the cip-cel operon is expressed at a higher level in the genetically modified Clostridium acetobutylicum than in the corresponding non-genetically modified Clostridium acetobutylicum.

[0017] The present invention also relates to a method for producing a target commodity chemical from lignocellulosic biomass, comprising culturing the genetically modified Clostridium acetobutylicum of the present invention on a suitable culture medium comprising lignocellulosic biomass as a major carbon source, and recovering the target commodity chemical from the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE of CipA:Cel48A and CipA:Cel48A_E61Q complex purified from C. acetobutylicum strains SC2 and SC3, respectively, by cellulose affinity chromatography. M: Molecular weight marker. E61Q:CipA:Cel48A_E61Q. Cel48A:CipA:Cel48A.

[0019] Figure 2 . SDS-PAGE of 30-fold concentrated supernatants from strain SC1 and strain Pthl-SAFA. M: molecular weight marker.

[0020] Figure 3 . SDS-PAGE of cellulosomes purified from strains SC1 and SC4.

[0021] Figure 4. A+B) shows the consumption rate and products produced by strain SC4 grown on PASC or AECC. C) Comparison of AECC consumption rates of strains SC1 and SC4.

[0022] Figure 5 . Comparison of AECC consumption rates between strains SC4 and SC5.

[0023] Figure 6 . SDS-PAGE of cellulosomes purified from strains SC4 and SC6. M: molecular weight marker.

[0024] Figure 7 . Comparison of AECC consumption rates between strains SC4 and SC6.

[0025] Figure 8 . Comparison of AECC consumption rates between strains SC4 and SC7.

[0026] Fig. 9 . SDS-PAGE of concentrated supernatants and purified cellulosomes from strains SC4 and SC6.

[0027] Fig.10 . SDS-PAGE of concentrated supernatants of strains SC9 and SC10.

[0028] Fig.11 . SDS-PAGE of purified cellulosomes from strains SC9 and SC10.

[0029] Fig.12 . Comparison of AECC consumption rates of strains SC8, SC9, and SC10.

[0030] Fig.13 . Comparison of AECC consumption rates between strain SC10 and Clostridium cellulophagus.

[0031] Fig.14 .Comparison of crystalline cellulose consumption rates of strains SC1, SC9, and SC10. DETAILED DESCRIPTION

[0032] General Definition

[0033] The term "commodity chemical" refers to chemicals in large volumes.

[0034] "Clostridium acetobutylicum" or "C. acetobutylicum" is known in the art and refers to a strain of Clostridium bacteria that produces acetone, butanol and ethanol and has a megaplasmid ( et al., 2001). "Genetically modified Clostridium acetobutylicum" refers to Clostridium acetobutylicum whose genome has been altered in the laboratory using genetic engineering techniques to facilitate the expression of a desired physiological trait.

[0035] When referring to the genetically modified Clostridium acetobutylicum of the present invention having an overexpressed operon or gene, "the corresponding non-genetically modified Clostridium acetobutylicum" refers to a Clostridium acetobutylicum strain having the same genome as the genetically modified Clostridium acetobutylicum of the present invention, except that there is no genetic modification inserted in the genome of the genetically modified Clostridium acetobutylicum of the present invention for overexpressing the operon or gene.

[0036] As used herein, when used to define products, compositions, and methods, the terms "comprising" (and any form of comprising, such as "comprising" and "including"), "having" (and any form of having, such as "having" and "having"), "including" (and any form of including, such as "including" and "including"), or "containing" (and any form of containing, such as "containing" and "containing") are open ended and do not exclude additional, unrecited elements or method steps. "Consisting essentially of" means excluding any other components or steps of significant significance. "Consisting of" means excluding more than trace amounts of other components or steps.

[0037] A "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of its associated gene or operon. In contrast, an "inducible promoter" refers to a regulated promoter that is active only in response to a specific stimulus.

[0038] "Operon" refers to a functional unit of DNA containing a cluster of genes under the control of a single promoter.

[0039] A gene is said to be "overexpressed" in a genetically modified Clostridium acetobutylicum when the gene is expressed at a level in the genetically modified Clostridium acetobutylicum that is greater than the expression level of the cip-cel operon in the corresponding non-genetically modified Clostridium acetobutylicum.

[0040] An "operon" is said to be "overexpressed" in a genetically modified Clostridium acetobutylicum when at least one gene, and preferably every gene, of the operon is expressed at a higher level in the genetically modified Clostridium acetobutylicum than in the corresponding non-genetically modified Clostridium acetobutylicum.

[0041] A native promoter of a gene or operon is said to be "replaced" by another promoter when the nucleic acid sequence of the native promoter is removed from the C. acetobutylicum genome and the nucleic acid sequence of another promoter is inserted into the C. acetobutylicum genome and operably linked to the gene or operon so as to direct its expression.

[0042] A native gene is said to be "replaced" by another gene at least when the protein-coding nucleic acid sequence of the gene is replaced by the protein-coding nucleic acid sequence of another gene. The regulatory sequence (especially the promoter) of the native gene may or may not be replaced by the regulatory sequence of another gene.

[0043] Unless otherwise indicated, all NCBI reference sequences (NCBI RefSeq) or Genbank accession numbers cited in this article include a version number (for RefSeq or Genbank accession number X, version n will be written as Xn) to fully define the exact sequence. Sequences are annotated as of September 16, 2022.

[0044] Genetically modified Clostridium acetobutylicum is able to grow on lignocellulosic biomass

[0045] The present invention relates for the first time to a genetically modified Clostridium acetobutylicum capable of growing on lignocellulosic biomass, wherein the cip-cel operon is overexpressed, and the expression level of each gene of the cip-cel operon in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

[0046] Clostridium acetobutylicum

[0047] Preferred strains of Clostridium acetobutylicum that can be genetically modified in the context of the present invention are those known for the production of solvents and other bulk chemicals (with or without genetic modification), such as Clostridium acetobutylicum ATCC 824, Clostridium acetobutylicum DSM 1731 and Clostridium acetobutylicum ATCC 4259, in particular Clostridium acetobutylicum ATCC 824 used in the experimental part.

[0048] The genome of Clostridium acetobutylicum ATCC 824 has been sequenced and annotated and is published under NCBI reference sequence NC_003030.1. The genome of Clostridium acetobutylicum ATCC 1731 has been sequenced and annotated and is published under NCBI reference sequence NC_015687.1.

[0049] Ability to grow on lignocellulosic biomass

[0050] The genetically modified Clostridium acetobutylicum of the present invention is capable of growing on lignocellulosic biomass.

[0051] Lignocellulosic biomass

[0052] "Lignocellulosic biomass" refers to any carbon source composed primarily of cellulose, hemicellulose, and lignin. Lignocellulosic biomass is the most abundant available feedstock on Earth for the production of biofuels (ethanol or butanol), so it is highly desirable to provide bacteria that can feed on lignocellulosic biomass and produce such biofuels.

[0053] Prior to fermentation with the genetically modified Clostridium acetobutylicum of the invention, the lignocellulosic biomass is preferably pretreated by one of a number of pre-established methods, including steam explosion, ammonia fiber expansion, alkaline extraction (e.g., alkaline extracted shelled corncobs, also known as "AECC", a commonly used model lignocellulosic biomass (Sudha Rani, Swamy and Seenayya 1998, 22)), organosolv treatment, or sulfite treatment (Zhao et al., 2022).

[0054] However, crystalline cellulose can also be used. "Crystalline cellulose" as used herein refers to type I cellulose, which is a natural cellulose found in most plant cell walls and structures, consisting of glucose units connected by 1-4β glycosidic bonds. Crystalline cellulose especially includes microcrystalline cellulose (abbreviated as "MCC"), such as commercially available Avicel PH products. When an organism is able to utilize crystalline cellulose as a carbon source and fully degrade it, it is generally considered that the organism is truly cellulose decomposing (cellulolytic) (see, for example, Koeck et al., Genomics of cellulolytic bacteria. Curr Opin Biotechnol. 2014. 29: 171-83).

[0055] Crystalline cellulose can be used as a substrate in particular when a genetically modified Clostridium acetobutylicum is used in which at least the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, an additional copy of the xynB gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, an additional copy of the cipA gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted (e.g., strain SC10). Therefore, strain SC10 can be considered a true cellulolytic organism.

[0056] Ability to grow on lignocellulosic biomass

[0057] In vitro, the ability of growing on lignocellulosic biomass can be measured and quantified by using a suitable culture medium comprising lignocellulosic biomass. For example, the acetobutylic acid clostridium of genetic modification is cultivated in a suitable culture medium comprising lignocellulosic biomass, and the amount of the lignocellulosic biomass consumed in the monitoring culture medium is monitored at least in 3 days. Optionally, the amount of one or more catabolites produced by lignocellulosic biomass (such as butyrate, acetate, butanol, ethanol ... depends on specific test strains) can also be monitored in the culture medium. If the consumption of interested lignocellulosic biomass is detected (optionally detecting the generation of at least one catabolite), the acetobutylic acid clostridium can grow on lignocellulosic biomass.

[0058] The expression "suitable medium" refers to a medium suitable for use with the genetically modified Clostridium acetobutylicum of the present invention, which is well known to those skilled in the art. Suitable medium known for culturing Clostridium acetobutylicum includes, but is not limited to, Clostridium Growth Medium (CGM) medium (Dusséaux et al., 2013), 2XYT (Oultram et al., 1988), minimal synthetic medium (MS, Soni et al., 1987), and Clostridium Basal Medium (CBM, see O'BRIEN et al., 1971).

[0059] To measure the ability to grow on lignocellulosic biomass, the lignocellulosic biomass preferably has been pretreated by one of the many established methods disclosed above.

[0060] Overexpression of the cip-cel operon

[0061] The genetically modified Clostridium acetobutylicum of the present invention has been genetically modified so that the cip-cel operon is overexpressed, ie, its expression level is higher than the expression level of the cip-cel operon in the corresponding non-genetically modified Clostridium acetobutylicum.

[0062] cip-cel operon

[0063] The C. acetobutylicum "cip-cel operon" or "cip-cel cluster" refers to the C. acetobutylicum operon comprising the genes in Table 1 below:

[0064] Table 1. Description of the genes contained in the cip-cel operon.

[0065]

[0066]

[0067] The nucleic acid sequence of the entire cip-cel operon (excluding the promoter region) corresponds to positions 1043161 to 1063760 of NC_003030.1.

[0068] All genes of the cip-cel operon showed linked transcription, under the control of the same promoter corresponding to positions 1040257 to 1043160 of NC_003030.1.

[0069] In the context of the present invention, the cel48A gene preferably encodes the native Cel48A protein of Clostridium acetobutylicum, i.e., the protein having the amino acid sequence WP_010964229.1. Preferably, the genetically modified Clostridium acetobutylicum comprises the native cel48A gene corresponding to the nucleic acid sequence of positions 1047700 to 1049880 of NC_003030.1. In one embodiment, all genes of the cip-cel operon are not genetically modified. In this case, the nucleic acid sequences of the genes of the cip-cel operon are as shown in Table 1 above.

[0070] Overexpression of the cip-cel operon

[0071] Overexpression of the cip-cel operon resulted from genetic modification of Clostridium acetobutylicum.

[0072] In order to overexpress the cip-cel operon, several types of genetic modifications can be performed. Specifically, the genetically modified Clostridium acetobutylicum having an overexpressed cip-cel operon of the present invention can be:

[0073] a) the native promoter of the cip-cel operon has been genetically modified, or

[0074] b) Additional copies of the cip-cel operon have been inserted into the C. acetobutylicum genome.

[0075] In a preferred embodiment, since all genes of the cip-cel operon are under the control of the same promoter, the promoter can be genetically modified to increase the expression of the operon genes. Several genetic modifications of the native promoter of the cip-cel operon can also be used here:

[0076] a1) The native promoter of the cip-cel operon could be replaced by a stronger promoter, or

[0077] a2) The native promoter of the cip-cel operon can be mutated.

[0078] The nucleic acid sequence of the native promoter of the cip-cel operon in Clostridium acetobutylicum is known in the art and corresponds to positions 1040257 to 1043160 of NC_003030.1.

[0079] Strong bacterial promoters are known in the art and option a1) above is a preferred embodiment, where the native promoter of the cip-cel operon can be replaced by a stronger promoter.

[0080] A "stronger promoter" refers to a promoter that is capable of inducing a higher level of expression of a given operon or gene than the native promoter. The stronger promoter is preferably constitutive, although a strong inducible promoter may also be used, provided that the promoter can be induced to express the cip-cel operon when the cells are grown on lignocellulosic biomass. Examples of constitutive promoters that are stronger than the native promoter of the C. acetobutylicum cip-cel operon include, but are not limited to, the promoters of the C. acetobutylicum thlA gene, the ptb gene, the crt gene, and the gapC gene.

[0081] The nucleic acid sequences of the above genes and their promoters are known in the art, as shown in Table 2 below.

[0082] Table 2. Relevant information about strong promoters.

[0083]

[0084]

[0085] In a particularly preferred embodiment, the native promoter of the cip-cel operon has been replaced by the promoter of the C. acetobutylicum thlA gene.

[0086] The native promoter of the cip-cel operon can be replaced with a stronger promoter using conventional genetic techniques, including homologous recombination-based replacement techniques that are effective in Clostridium (as disclosed in WO2008040387), ACE or CRISPR / Cas9 editing of the Clostridium acetobutylicum genome (see Ehsaan et al., ACE editing in 2016 and CRISPR / Cas9 editing in Wilding-Steele et al., 2021). Detailed technical information on how to perform the replacement is provided in Example 1 below.

[0087] However, the natural promoter may also be maintained in a mutated form that allows for a higher expression of the cip-cel operon. In particular, a counter-control element present in the promoter, such as a catabolite response element (CRE), may be deleted from the natural promoter.

[0088] In this regard, it should be noted that the cip-cel operon of C. acetobutylicum has been found to be highly similar to the cip-cel operon of Clostridium cellulolyticum (see Sabathé, F. & al. 2002), the promoter of the cip-cel operon of C. cellulolyticum has been studied, in which a CRE was found, the deletion of which resulted in a higher expression of the cip-cel operon (see Abdou et al., 2007). In the context of the present invention, similar genetic modifications of the native promoter of the cip-cel operon of C. acetobutylicum can be considered.

[0089] Alternatively, or in combination with genetic modification of the native promoter of the cip-cel operon of C. acetobutylicum, additional copies of the cip-cel operon may be inserted into the C. acetobutylicum genome.

[0090] In this case, the extra copies of the cip-cel operon are preferably placed under the control of a stronger promoter than the native promoter of the C. acetobutylicum cip-cel operon (see above for exemplary stronger promoters).

[0091] Additional copies of the cip-cel operon can be inserted into the C. acetobutylicum genome by conventional genetic techniques, including use of the publicly available ACE (Ehsaan et al., 2016) or CRISPR / Cas9 editing of the C. acetobutylicum genome (see Wilding-Steele et al., 2021).

[0092] The additional copy of the cip-cel operon inserted into the genome of C. acetobutylicum may or may not contain a promoter that is stronger than the native promoter of the cip-cel operon (which is operably linked to the cip-cel operon), depending on the position where the additional copy is inserted. When the additional copy of the cip-cel operon is inserted at a position under the control of the native promoter of the genome of C. acetobutylicum, a promoter is not required in the inserted sequence (see the xynB gene in Example 8 below). If not, the inserted sequence should further contain a promoter that is stronger than the native promoter of the cip-cel operon, which is operably linked to the cip-cel operon.

[0093] Overexpression of other genes

[0094] To further improve growth on lignocellulosic biomass, the genetically modified Clostridium acetobutylicum of the present invention can be further genetically modified to overexpress other genes that increase cellulosic activity.

[0095] Such a gene increasing cellulosomal activity may in particular be selected from cellulases and xylanases located outside the cip-cel operon.

[0096] Specifically, the genetically modified Clostridium acetobutylicum of the present invention can be further genetically modified so that one or more genes in Table 3 below are overexpressed, so that their expression levels in the genetically modified Clostridium acetobutylicum are higher than their expression levels in the corresponding non-genetically modified Clostridium acetobutylicum.

[0097] Table 3. Clostridium acetobutylicum genes that can be further overexpressed in the genetically modified Clostridium acetobutylicum of the present invention.

[0098]

[0099] Optional overexpression of cel9X gene

[0100] In a preferred embodiment, the genetically modified Clostridium acetobutylicum of the present invention has been further genetically modified such that the cel9X gene is overexpressed, and its expression level in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

[0101] Overexpression of the cel9X gene is due to genetic modification of Clostridium acetobutylicum.

[0102] In order to overexpress the cel9X gene, several types of genetic modifications can be performed. Specifically, the genetically modified Clostridium acetobutylicum of the present invention having an overexpressed cel9X gene can:

[0103] a) the native promoter of the cel9X gene has been genetically modified, or

[0104] b) An extra copy of the cel9X gene has been inserted into the C. acetobutylicum genome.

[0105] In one embodiment, the natural promoter of the cel9X gene can be genetically modified to increase the expression of the cel9X gene. Several genetic modifications of the natural promoter of the cel9X gene can also be used here:

[0106] a1) The natural promoter of the cel9X gene can be replaced by a stronger promoter, or

[0107] a2) The native promoter of the cel9X gene can be mutated.

[0108] The stronger promoter is preferably constitutive. Examples of constitutive promoters that are stronger than the native promoter of the C. acetobutylicum cel9X gene include, but are not limited to, the promoters of the C. acetobutylicum thlA gene, ptb gene, crt gene, and gapC gene, as shown in Table 2 above.

[0109] The native promoter of the cel9X gene can be replaced with a stronger promoter using conventional genetic techniques (see cip-cel operon above and Example 3 below for a specific protocol).

[0110] Additional copies of the cel9X gene can be inserted into the C. acetobutylicum genome by conventional genetic techniques (see above for the general description of the cip-cel operon and below for the description of the xynB gene in Example 8).

[0111] Overexpression of the optional cel5Y gene

[0112] Alternatively or in combination with further overexpression of the cel9X gene, the genetically modified Clostridium acetobutylicum of the present invention can be further genetically modified such that the cel5Y gene is overexpressed, the expression level of which in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

[0113] Overexpression of the cel5Y gene is due to genetic modification of Clostridium acetobutylicum.

[0114] In order to overexpress the cel5Y gene, several types of genetic modifications can be performed. Specifically, the genetically modified Clostridium acetobutylicum having an overexpressed cel5Y gene of the present invention can be:

[0115] a) The natural promoter of the cel5Y gene is genetically modified, preferably:

[0116] a1) The native promoter of the cel5Y gene has been replaced by a stronger promoter, or

[0117] a2) The native promoter of the cel5Y gene has been mutated.

[0118] b) an extra copy of the cel5Y gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cel5Y gene,

[0119] The stronger promoter is preferably constitutive. Examples of constitutive promoters that are stronger than the native promoter of the C. acetobutylicum cel5Y gene include, but are not limited to, the promoters of the C. acetobutylicum thlA gene, ptb gene, crt gene, and gapC gene, as shown in Table 2 above.

[0120] The native promoter of the cel5Y gene can be replaced with a stronger promoter using conventional genetic techniques (see general description above and specific protocols in Example 7 below).

[0121] Additional copies of the cel5Y gene can be inserted into the C. acetobutylicum genome by conventional genetic techniques (see above for a general description of the cip-cel operon and below for a description of the insertion of additional copies of the xynB gene in Example 8).

[0122] Overexpression of the optional xynB gene

[0123] Alternatively or in combination with further overexpression of the cel9X gene and / or the cel5Y gene, the genetically modified Clostridium acetobutylicum of the present invention may be further genetically modified such that the xynB gene is overexpressed, the expression level of which in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

[0124] Overexpression of the xynB gene is due to genetic modification of C. acetobutylicum.

[0125] In order to overexpress the xynB gene, several types of genetic modifications can be performed. Specifically, the genetically modified Clostridium acetobutylicum with an overexpressed xynB gene of the present invention can be:

[0126] a) an extra copy of the xynB gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the xynB gene,

[0127] b) the natural promoter of the xynB gene is genetically modified, preferably:

[0128] b1) The native promoter of the xynB gene has been replaced by a stronger promoter, or

[0129] b2) The native promoter of the xynB gene has been mutated.

[0130] The stronger promoter is preferably constitutive. Examples of constitutive promoters that are stronger than the native promoter of the C. acetobutylicum xynB gene include, but are not limited to, the promoters of the C. acetobutylicum thlA gene, ptb gene, crt gene, and gapC gene, as shown in Table 2 above.

[0131] The native promoter of the xynB gene can be replaced with a stronger promoter using conventional genetic techniques (see the general description above and the cip-cel operon, cel9X gene and cel5Y gene of Examples 1, 3 and 7 below).

[0132] Additional copies of the xynB gene can be inserted into the C. acetobutylicum genome by conventional genetic techniques (see above for a general description of the cip-cel operon and below in Example 8 for a specific protocol for the insertion of additional copies of the xynB gene).

[0133] Since the xynB gene is located on a megaplasmid of C. acetobutylicum, the additional copy of the xynB gene inserted in the genome of C. acetobutylicum is preferably under the control of a stronger promoter than the native promoter of the xynB gene.

[0134] The optional cipA gene is further overexpressed, resulting in the production of cipA scaffold protein and the production of cellulosomes The ratio of cellulase increased.

[0135] For optimal cellulolytic activity, all cellulases containing anchor domains should be linked to a scaffold protein via their anchor-adhesin domains. In the case of the present application, the scaffold protein CipA of Clostridium acetobutylicum contains five adhesin domains, which means that there should be one CipA protein for five anchor-containing cellulases.

[0136] When overexpressing the entire cip-cel operon, the inventors found that the ratio of scaffold CipA protein to anchor-containing cellulase was not optimal for cellulolytic activity and further overexpression of the cipA gene encoding the scaffold protein allowed further improvement of the ratio and cellulolytic activity.

[0137] Therefore, as an alternative, or in combination with further overexpression of one or more of the cel9X, cel5Y and xynB genes, the genetically modified Clostridium acetobutylicum of the present invention can be further genetically modified such that the cipA gene is further overexpressed relative to the other genes of the cip-cel operon. Such modification preferably results in an increased ratio of the CipA scaffold protein produced to the cellulosome cellulase produced. This, in turn, preferably results in higher amounts of cellulosomes and lower amounts of free cellulase.

[0138] The cipA gene is a gene in the cip-cel operon, so overexpression of the cip-cel operon leads to overexpression of the cipA gene.

[0139] However, compared with other genes in the cip-cel operon, the cipA gene can be further overexpressed due to additional genetic modifications.

[0140] In order to further overexpress the cipA gene compared to other genes of the cip-cel operon, several types of genetic modifications can be performed. Specifically, the genetically modified Clostridium acetobutylicum of the present invention having a cipA gene that is further overexpressed compared to other genes of the cip-cel operon can:

[0141] a) an extra copy of the cipA gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cipA gene,

[0142] b) The ribosome binding site (abbreviated as "RBS") of the cipA gene is changed to a stronger RBS. Publicly available tools can be used to optimize stronger RBS sequences for specific species and protein sequences, such as the RBS calculator of the Salis laboratory (Reis AC, Salis HM. An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models. ACS Synth Biol. 2020. 9 (11): 3145-3156.).

[0143] c)

[0144] Stabilizing the mRNA of the cip-cel operon in this way specifically improves the stability of the transcript encoding cipA, as shown by Xu et al. Cellulosome stoichiometry in Clostridium cellulolyticum isregulated by selective RNA processing and stabilization. Nat Commun. 2015. 6: 6900. This paper demonstrated that various stem-loops or 3'UTR' can stabilize the mRNA transcript of the cip-cel operon of Archaea cellulolytica by protecting it from exonuclease degradation. In addition, they showed that this protection is related to the free folding energy (ΔG) of the stem-loop. They determined the ΔG of the stem-loop of the cip-cel operon of a variety of different species, including Clostridium acetobutylicum. The ΔG of the stem-loop between cipA and cel48A is relatively high, only -20, while the stem-loop between cipC and cel48F of Archaea cellulolytica is -33.8. The cip-cel operon of C. acetobutylicum was modified by modifying the stem-loop between cipA and cel48A to a stem-loop with a lower ΔG, for example, the stem-loop between cipC and cel48F of A. cellulolyticus, resulting in increased stability of the mRNA transcript and thus increased amounts of cipA produced.

[0145] In a preferred embodiment, in order to further overexpress the cipA gene compared to the other genes of the cip-cel operon, an additional copy of the cipA gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cipA gene.

[0146] Additional copies of the cipA gene can be inserted into the C. acetobutylicum genome by conventional genetic techniques (see above for a general description of the cip-cel operon and Example 10 below for a specific protocol for the insertion of additional copies of the cipA gene).

[0147] Optionally, inactivation of genes encoding extracellular proteases

[0148] As an alternative, or in combination with further overexpression of one or more of the cel9X, cel5Y and xynB genes, or in combination with further overexpression of one or more of the cel9X, cel5Y, xynB and cipA genes, the genetically modified Clostridium acetobutylicum of the present invention may be further genetically modified by inactivating genes encoding extracellular proteases in order to improve growth on lignocellulosic biomass. This is because extracellular proteases can degrade cellulosomes, thereby reducing the amount of cellulosomes and limiting their ability to digest lignocellulosic biomass.

[0149] The extracellular protease is preferably selected from nrpE, CA_C0746, CA_C0625 and CA_C2695. More preferably, the extracellular protease is encoded by the gene nrpE because the extracellular protease is expressed the most. In other preferred embodiments, several extracellular proteases can be inactivated in the same strain to further reduce the number of extracellular proteases, thereby further reducing cellulosome degradation. In this case, the inactivated extracellular protease more preferably comprises an extracellular protease encoded by the gene nrpE and one or more extracellular proteases encoded by the genes CA_C0746, CA_C0625 and CA_C2695.

[0150] Table 4 below provides information on genes encoding extracellular proteases that are deleted in the genetically modified Clostridium acetobutylicum of the present invention to enhance growth on lignocellulosic biomass.

[0151] Table 4. Clostridium acetobutylicum genes encoding extracellular proteases that may be deleted in the genetically modified Clostridium acetobutylicum of the present invention.

[0152]

[0153] Inactivation of extracellular proteases is due to genetic modification of C. acetobutylicum.

[0154] Inactivation of extracellular proteases can be achieved by any technique known in the art, including:

[0155] a) partial or complete deletion of genes encoding extracellular proteases (particularly nrpE), and

[0156] b) Insertion of inactivating mutations in genes encoding extracellular proteases, particularly nrpE.

[0157] Examples of inactivating mutations include insertion of an intron sequence, or insertion of a sequence encoding another protein into a sequence encoding an extracellular protease (particularly nrpE), or mutations in the active site of an extracellular protease (particularly nrpE) that result in reduced or inhibited protease activity.

[0158] In the case of the extracellular protein encoded by the nrpE gene, the active site includes amino acids 379E and 478H. Thus, inactivation can be obtained by replacing 379E and / or 478H with another amino acid, preferably another different type of amino acid.

[0159] Partial or complete deletion of genes encoding extracellular proteases (particularly nrpE) is the easiest way to eliminate protease activity and is therefore preferred, and can be performed by conventional genetic techniques, including efficient replacement techniques in Clostridium based on homologous recombination, as disclosed in WO2008040387 (no sequence between the two homologous arms) or ACE or CRISPR / Cas9 editing of the Clostridium acetobutylicum genome (see, ACE by Ehsaan et al. 2016 and CRISPR / Cas9 editing by Wilding-Steele et al., 2021).

[0160] In case of a partial deletion, preferably at least 50% of the coding sequence is removed, more preferably at least 80% is removed.

[0161] Inactivating mutations can be inserted using conventional mutagenesis techniques, depending on the type of inactivating mutation.

[0162] Preferred genetically modified Clostridium acetobutylicum

[0163] The genetically modified Clostridium acetobutylicum of the present invention may combine several of the genetic modifications disclosed above to improve growth on lignocellulosic biomass.

[0164] Specifically, the preferred genetically modified Clostridium acetobutylicum strain of the present invention can be selected from:

[0165] a) A genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene.

[0166] An example of such a strain is the Clostridium acetobutylicum strain SC1 obtained in Example 1 below.

[0167] b) A genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene and the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene.

[0168] An example of such a strain is the Clostridium acetobutylicum strain SC4 obtained in Example 3 below.

[0169] c) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene,

[0170] An example of such a strain is the Clostridium acetobutylicum strain SC6 obtained in Example 7 below.

[0171] d) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and an additional copy of the xynB gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene,

[0172] An example of such a strain is the Clostridium acetobutylicum strain SC7 obtained in Example 8 below.

[0173] e) A genetically modified Clostridium acetobutylicum, wherein the natural promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the natural promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted.

[0174] An example of such a strain is the Clostridium acetobutylicum strain SC5 obtained in Example 6 below.

[0175] f) A genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted.

[0176] An example of such a strain is the Clostridium acetobutylicum strain SC8 obtained in Example 9 below.

[0177] g) A genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, an extra copy of the xynB gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted.

[0178] An example of such a strain is the Clostridium acetobutylicum strain SC9 obtained in Example 9 below.

[0179] h) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, an extra copy of the xynB gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, an extra copy of the cipA gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted.

[0180] An example of such a strain is the Clostridium acetobutylicum strain SC10 obtained in Example 10 below.

[0181] Optional further genetic processing for production of specific target commodity chemicals

[0182] Based on the genetic modifications known in the prior art to optimize the production of specific target chemicals of interest, in particular by attenuating and / or deleting and / or replacing specific genes to promote the metabolic pathways for the production of target bulk chemicals, the genetically modified Clostridium acetobutylicum of the present invention can be further genetically modified to increase the production of target bulk chemicals.

[0183] The bulk chemicals that can be produced by culturing the genetically modified Clostridium acetobutylicum of the present invention are known in the art, and are preferably selected from ethanol, butanol, glycerol, 1,2-propylene glycol, acetone, isopropanol, isobutylene, hydrogen, acetic acid and lactic acid, etc.

[0184] Non-limiting examples of additional modifications for optimizing the production of ethanol, butanol, and lactic acid are disclosed below, but the genetically modified Clostridium acetobutylicum of the present invention may also be further genetically modified to optimize the production of other target chemicals of interest.

[0185] Optimizing ethanol production

[0186] Suitable modifications to optimize ethanol production are known in the art.

[0187] Specifically, the genetically modified Clostridium acetobutylicum of the present invention can be further modified to optimize ethanol production by inactivating (including partial or complete deletion and insertion of inactivating mutations) lactate dehydrogenase (ldhA), thiolase (thlA) and hydrogenase (hydA) genes (see US20170240869A1, although the document does not mention the inactivation of the ldhA gene, it should be noted that the ldhA gene has been deleted in the strains in which thlA and hydA are inactivated).

[0188] Information on the thlA gene and the ptb gene has been provided in Table 2 above.

[0189] Relevant information on the use of the hydA gene and the ldhA gene for optimizing butanol production is provided in Table 5 below, which also includes information on the hbd gene and the buk gene.

[0190] Table 5. C. acetobutylicum genes can be further deleted or overexpressed in the genetically modified C. acetobutylicum of the invention to optimize the production of ethanol, butanol or lactate.

[0191]

[0192] The above modifications can be performed using conventional techniques (see US20170240869A1).

[0193] Optimizing butanol production

[0194] Suitable modifications to optimize butanol production are known in the art.

[0195] In particular, the genetically modified Clostridium acetobutylicum of the present invention can be further modified to optimize butanol production by inactivating (including partial or complete deletion and insertion of inactivating mutations) the ptb-buk operon, replacing the thlA gene of Clostridium acetobutylicum with the Escherichia coli atoB gene, replacing the hbd gene of Clostridium acetobutylicum with the Clostridium kluyveri hbd1 gene, and overexpressing the CA_C0764 gene (whose expression level is higher than that in the corresponding non-genetically modified Clostridium acetobutylicum) (see Nguyen et al., 2018; Foulquier et al., 2022).

[0196] The sequence of the E. coli atoB gene can be found in NC_000913.3 (positions 2326109 to 2327293).

[0197] The sequence of the C. kluyveri hbd1 gene can be found in CP000673.1 (positions 437268 to 438116).

[0198] The sequence of the C. acetobutylicum CA_C0764 gene corresponds to positions 886021 to 887256 in NC_003030.1.

[0199] These modifications can be made using conventional techniques (e.g., see Nguyen et al. 2018; Foulquier et al., 2022).

[0200] Optimizing lactic acid production

[0201] Suitable modifications to optimize lactate production are known in the prior art.

[0202] In particular, the genetically modified Clostridium acetobutylicum of the present invention can be further modified to optimize lactic acid production by inactivating (including partial or complete deletion and insertion of inactivating mutations) thiolase (thlA) and hydrogenase (hydA) genes, followed by removal of giant plasmids (see US20170240869A1, please note that in this document, the ldhA gene was first restored because the starting strain had already lost the ldhA gene). If the genetically modified Clostridium acetobutylicum of the present invention inactivates the ldhA gene, the gene should be restored.

[0203] In the context of the present invention, the term "curing" when used with a giant plasmid refers to the removal of the giant plasmid from cells containing the microorganism by a suitable method (e.g., by using Crispr-Cas9 to target a suitable sequence on the giant plasmid) (see also those disclosed in US20170240869A1)

[0204] Methods for producing targeted commodity chemicals from lignocellulosic biomass

[0205] The present invention also relates to a method for producing a target commodity chemical from lignocellulosic biomass, comprising culturing the genetically modified Clostridium acetobutylicum of the present invention in a suitable culture medium comprising lignocellulosic biomass as a major carbon source, and recovering the target commodity chemical from the culture medium.

[0206] Suitable culture media known for culturing Clostridium acetobutylicum include, but are not limited to, Clostridial Growth Medium (CGM) medium (Dusséaux et al., 2013), 2XYT (Oultram et al., 1988), Synthetic Minimal Medium (MS, Soni et al., 1987), and Clostridial Basal Medium (CBM, see O'BRIEN et al., 1971).

[0207] In the above methods, the lignocellulosic biomass is preferably pretreated by one of many pre-established methods, including steam explosion, ammonia fiber expansion, alkaline extraction (e.g., alkaline extracted dehulled corncob, also known as "AECC"), organic solvent treatment, or sulfite treatment (Zhao et al., 2022).

[0208] The bulk chemicals that can be easily produced by culturing the genetically modified Clostridium acetobutylicum of the present invention are known in the art, and are preferably selected from ethanol, butanol, glycerol, 1,2-propylene glycol, acetone, isopropanol, isobutylene, hydrogen, acetic acid, lactic acid, and the like.

[0209] Depending on the specific target bulk chemical, one skilled in the art will know which genetically modified Clostridium acetobutylicum of the present invention is more suitable.

[0210] The following examples are intended only to illustrate the present invention.

[0211] Example

[0212] Example 1: The natural Cel48A protein of Clostridium acetobutylicum is active

[0213] 1: Production of Cel48A in E. coli results in protein misfolding.

[0214] EP2436698A1 stated that Cel48A was not functional. We have demonstrated that Cel48A is in fact functional. In EP2436698A1, a sequence clone of Cel48A was purified from E. coli and showed very low activity on CMC and PASC and no activity on crystalline cellulose.

[0215] We later demonstrated that Cel48A purified in this way was not actually folded correctly, which may explain its lack of activity. Specifically, although Cel48A appeared soluble when initially purified, after ultracentrifugation (performed at 100,000 RPM), 100% of Cel48A was found to be in the insoluble fraction. This was not the case with Cel48F purified from Archaea cellulolyticus as a control.

[0216] 2: Expression of Cel48a in Clostridium acetobutylicum

[0217] The activity of Cel48A was determined by purifying Cel48A directly from Clostridium acetobutylicum. First, the natural promoter of the cip-cel operon was replaced with the constitutive thiolase (thlA or thl) promoter (pthl) to construct strain SC1. To this end, a previously described strategy (Wilding-Steele et al., 2021) was used, with CAS2ΔldhA as the base strain and plasmid pGRNA_pthl-cip-cel for genetic modification. The plasmid was constructed by Genewiz, using the expression cassette pGRNAJ23119Δupp as a template (Wilding-Steele et al., 2021), and the expression cassette was cut with SacI / EcoRI, and the synthetic sequence synthesized by Genewiz was inserted into it. The synthetic sequence of plasmid pGRNA_pthl-cip-cel is defined as SEQ ID NO: 1.

[0218] Secondly, a second copy of orfXp and a terminator were inserted between cel48A and cel5B to construct strain SC2, which produced a truncated transcript, thereby expressing only cipA, cel48A and orfXp. This was achieved using SC1 as the base strain and plasmid pGRNA_cel48A_T1T2. The plasmid was constructed by Genewiz, using the expression cassette pGRNAJ23119Δupp as a template, and the expression cassette was cut by SacI / EcoRI, and the synthetic sequence synthesized by Genewiz was inserted into it. The synthetic sequence of plasmid pGRNA_cel48A_T1T2 is defined as SEQ ID NO: 2.

[0219] Finally, a mutation was introduced in Cel48A, and the glutamic acid at position 66 was replaced with glutamine to construct strain SC3. This is the equivalent of Cel48F_E55Q, which has been shown to cause enzyme inactivation because E55 is a proton donor. This was achieved using SC2 as a base strain and using plasmid pGRNA_cel48A_E61Q. The plasmid was constructed by Genewiz, using expression cassette pGRNAJ23119Δupp as a template, and the expression cassette was cut by SacI / EcoRI, and the synthetic sequence synthesized by Genewiz was inserted therein. The synthetic sequence of plasmid pGRNA_Cel48A_E61Q is defined as SEQ ID NO:3.

[0220] 3: Catalytic properties of CipA:Cel48A and CipA:Cel48A_E61Q complexes

[0221] The CipA:Cel48A complex was purified from strains SC2 and SC3 by growth in MS medium (Soni et al., 1987) and then purified using cellulose affinity chromatography. The purity of the complex was verified by SDS-PAGE and proteomic analysis ( Figure 1 ).

[0222] The specific activity of CipA:Cel48A complex was studied using phosphoric acid swollen cellulose (PASC) and Avicel (crystalline cellulose) as substrates. The degradation activity was tracked for at least two hours. The results are summarized in Table 6 and compared with the activity of the homologous Cel48F known to have effective activity on crystalline cellulose (Reverbel-Leroy et al., 1997). The activity assay showed that the CipA:Cel48A complex was active on PASC and Avicel, while the CipA:Cel48A_E61Q complex showed very low activity (as expected). This shows that Cel48A is secreted in an active form by Clostridium acetobutylicum.

[0223] Table 6. Activity of the CipA:Cel48A complex and the CipA:Cel48A_E61Q complex on various substrates compared to the activity of the previously characterized GH48 cellulase from Archaea cellulolyticus.

[0224] Activity (mU / mg) Cel48A Cel48A_E61Q Cel48F PASC 25.7 2.7 130 Avicel 4.8 1.3 3.2

[0225] Example 2: Cel48A expression level is significantly higher than Cel48A_SAFA

[0226] The Clostridium acetobutylicum SAFA strain disclosed in EP2436698A1 (wherein cel48A has been replaced by cel48SAFA) was genetically modified by replacing the promoter of the cip-cel operon with the thiolase (thlA) promoter to construct the strain Clostridium acetobutylicum Pthl-SAFA.

[0227] Comparison of the expression levels of cellulosomal components in this strain and strain SC1 (which is identical except for the presence of the native cel48A gene replacement instead of the hybrid cel48SAFA gene) showed that Cel48SAFA was produced at significantly lower levels compared to native Cel48A ( Figure 2 ).

[0228] Example 3: Expression of cel9X in Clostridium acetobutylicum

[0229] The natural promoter of cel9X was changed to a thiolase promoter, strain SC1 was used as the base strain, and plasmid pGRNA_pthl-cel9X was used for modification to construct strain SC4. This plasmid was constructed by Genewiz, using the expression cassette pGRNAJ23119Δupp as a template (Wilding-Steele et al., 2021), and the synthetic sequence synthesized by Genewiz was inserted into it by cutting the expression cassette with SacI / EcoRI. The synthetic sequence of plasmid pGRNA_pthl-cel9X is defined as SEQ ID NO:4.

[0230] Analysis of the supernatant showed that, in addition to the proteins present in the cip-cel operon, CEL9X ( Figure 3 ).

[0231] Example 4: Catalytic activity of cellulosomes produced by recombinant Clostridium acetobutylicum

[0232] Cellulosomes from SC1, SC4, and Archaea cellulolyticus were purified using cellulose affinity chromatography.

[0233] Activity assays showed that cellulosomes from SC1, SC4 and A. cellulolyticus showed similar activities on PASC and Avicel (Table 7).

[0234] Table 7. Activity of purified cellulosomes from strains SC1 and SC4 on various substrates and compared with the activity of previously characterized cellulosomes from Archaea cellulolyticus.

[0235] Activity (mU / mg) SC1 SC4 Archaea cellulolytica PASC 281 403 368 Avicel 9.4 19.3 39

[0236] Example 5: Recombinant strains are able to grow on lignocellulosic biomass

[0237] The ability of strain SC4 to grow on lignocellulosic biomass was evaluated. Small-scale flask fermentation experiments were performed in which cells were grown in CGM medium containing 5 g / L PASC or 30 g / L AECC. SC4 was able to grow on both PASC and AECC ( Figure 4A and Figure 4B ).

[0238] Finally, strain SC4 was shown to grow faster on AECC than strain SC1, especially at the end of the culture, probably because the addition of Cel9X enabled this strain to degrade the more difficult cellulose ( Figure 4C ).

[0239] Example 6: Lack of extracellular proteases leads to improved growth on lignocellulosic biomass

[0240] nrpE is a gene encoding an extracellular protease that can degrade cellulosomal components. Strain SC4 was used as a base strain to delete the gene nrpE. Plasmid pGRNA_ΔnrpE was used for modification to construct strain SC5. This plasmid was constructed by Genewiz, using the expression cassette pGRNAJ23119Δupp as a template (Wilding-Steele et al., 2021). The expression cassette was cut with SacI / EcoRI, and the synthetic sequence synthesized by Genewiz was inserted into it. The synthetic sequence of plasmid pGRNA_ΔnrpE is defined as SEQ ID NO:5.

[0241] The growth of strains SC4 and SC5 on AECC showed that strain SC5 grew significantly faster ( Figure 5 In addition, higher amounts of cellulosomes were produced in strain SC5 compared to strain SC4 (data not shown).

[0242] Example 7: Expression of cel5Y

[0243] Cel5Y is another cellulosomal cellulase that we found to be able to bind cellulosomes to the cell wall. Based on this, we hypothesized that its overexpression might further improve growth on lignocellulosic biomass.

[0244] cel5Y was overexpressed by changing its native promoter to a strong thiolase promoter (pthl). Strain SC4 was used as the base strain for this modification. Plasmid pGRNA_pthl_cel5Y was used for modification to construct strain SC6. This plasmid was constructed by Genewiz using the expression cassette pGRNAJ23119Δupp as a template (Wilding-Steele et al., 2021). The expression cassette was cut with SacI / EcoRI and the synthetic sequence synthesized by Genewiz was inserted into it. The synthetic sequence of plasmid pGRNA_pthl_Cel5Y is defined as SEQ ID NO:6.

[0245] Purification of cellulosomes from SC4 and SC6 ( Figure 6 ), showing that cellulosomes purified from SC6 had higher activity levels on PASC and Avicel (Table 8).

[0246] Table 8. Activity of cellulosomes purified from strains SC4 and SC6 on various substrates.

[0247] Activity (mU / mg) SC4 SC6 PASC 403 1333.3 Avicel 19.3 25.6

[0248] In addition, strains SC4 and SC5 were grown on AECC, and strain SC6 showed faster growth ( Figure 7 ).

[0249] Example 8: Expression of xynB leads to improved growth on lignocellulosic biomass

[0250] Since the goal was to grow on pretreated lignocellulose containing cellulose and hemicellulose, it was hypothesized that co-expression of xylanases would increase the growth rate on pretreated lignocellulose.

[0251] XynB, one of the native xylanases of C. acetobutylicum, was overproduced by cloning an extra copy of the xynB gene downstream of the thiolase gene. Strain SC4 was used as the base strain for this modification. Plasmid pMTL_JH16-xynB was used for modification to construct strain SC7. The plasmid was constructed using plasmid pMTL_JH16 (GenBank Accession No. HQ875757.1) as a template, using oligo 1 and 2 (oligo 1 = XynB-not1: agcggccgcAATTAATTATTAAATAAA (SEQ ID No: 7); oligo 2 = XynB-Nhe1: gctagcaaagCTAATGTGATGCT (SEQ ID NO: 8)) for PCR, using gDNA of C. acetobutylicum as a template. The expression cassette and PCR product were digested with Not1 / Nhe1 and ligated together.

[0252] SC4 and SC7 were grown on AECC, showing that strain SC7 grew faster ( Figure 8 ).

[0253] Example 9: Construction and characterization of strains SC8 and SC9 by overexpression of the cip-cel operon, cel9X, cel5Y (and optionally xynB) and deletion of the extracellular protease NrpE

[0254] Strain SC8 was constructed using strain SC6 as a base strain. Strain SC8 was constructed using plasmid pGRNA_ΔnrpE with modifications (see Example 6).

[0255] Strain SC8 was used as a base strain to construct strain SC9. Strain SC9 was constructed using plasmid pMTL_JH16-xynB with modifications (see Example 8).

[0256] Example 10: Addition of a second copy of the cipA scaffold protein gene results in improved growth on lignocellulosic biomass

[0257] Initial analysis using SDS-PAGE showed that the cellulosomes were likely saturated, meaning that the proteins containing the anchor were in excess relative to the cohesin domain, meaning that not all cellulases were able to bind to the scaffold protein CipA. This was also shown by the significantly reduced intensity of the band corresponding to Cel48A in the purified cellulosome fraction compared to the supernatant. It is important to note that high levels of Cel9X and Cel5Y may not have bound to the cellulosomes but rather co-purified with them because they contain the cellulose binding domain, while Cel48A does not ( Fig. 9 ). This could result in reduced activity and stability of the cellulases, as binding of cellulases to scaffold proteins has been shown previously to increase their activity and stability.

[0258] To increase the production of CipA, a second copy of cipA was introduced downstream of the thiolase gene in the same operon as xynB. This modification was performed in strain SC8 (constructed by deleting nrpE in strain SC6) using plasmid pMTL_JH16-cipA-xynB, resulting in strain SC10. SC9 was also constructed as a control by using plasmid pMTL_JH16-xynB. Plasmid pMTL_JH16-cipA-xynB was constructed using plasmid pMTL_JH16-xynB as a template, using the following primers for PCR: forward primer cipa-not1_fw: GCAGATGGCGGCCGCggcccagaatttaaaaggagg (SEQ ID NO: 11) and reverse primer cipa-not1-rev: caacaaatggaaaaataactgttgaataaGCGGCCGCatttc (SEQ ID NO: 12), and gDNA of Clostridium acetobutylicum was used as a template. The expression cassette and PCR product were digested with Not1 and ligated together.

[0259] Strains SC9 and SC10 were grown in triplicate in MS-cellobiose to an OD600 of 1.5. The supernatant was then collected by centrifugation and subsequently concentrated using ultrafiltration (Vivaspin Turbo 15 10,000MWCO) and dialyzed in sodium acetate buffer (20mM sodium acetate, 10mM calcium chloride, 100mM sodium chloride, pH 5.5). The protein concentration was determined using the Bradford method and the protein concentration was standardized to 0.1mg / ml. The supernatant was then analyzed by SDS-PAGE and quantitative proteomics analysis. SDS-PAGE showed that the band at 180KDa (possibly corresponding to CipA) of strain SC10 showed a stronger band intensity than that of SC9 ( Fig.10). Quantitative proteomic analysis was performed using a TripleTOF 6600 mass spectrometer (Sciex). Sequential window acquisition (SWATH) of all theoretical spectra and additional data processing were performed using DIA-NN. In order to be able to compare the amounts of each protein produced, the molar amount of each protein was determined and the molar percentage was then calculated by dividing the molar amount of each of the proteins by the molar amount of all supernatant proteins. Therefore, proteomic analysis showed that the amount of cipA in strain SC10 increased nearly 3 times compared to SC9 (Table 9). Data analysis showed that the cohesin / anchor ratio was 0.54 for SC9 and 1.54 for strain SC10 (the ratio was calculated by determining the number of cohesin domains (the number of moles of CipA multiplied by 5) divided by the total number of anchor-containing proteins). This confirms that strain SC10 has sufficient CipA production, which is not the case for strain SC9.

[0260] Table 9. Mole percent of cellulosic proteins found in the supernatants of strains SC9 and SC10

[0261] Cellulosomal proteins SC9 SC10 CipA 0.79 2.36 Cel48A 2.81 2.87 Cel9X 1.40 1.84 Cel5Y 1.59 1.46 Cel5B 0.83 0.70 Cel9C 0.23 0.18 f 0.00 0.00 Cel44D 0.08 0.09 Cel9E 0.02 0.06 Cel9F 0.04 0.11 man5G 0.21 0.27 cel74h 0.11 0.08 Total cellulosomal 8.12 10.01 Total anchor protein 7.33 7.66 Total adhesin 3.94 11.78 Anchor / adhesin ratio 0.54 1.54

[0262] Cellulosomes were purified from SC9 and SC10 ( Fig.11 ), the activity levels of cellulosomes purified from SC10 on Avicel were higher than those purified from SC9 (see Table 10), indicating that the addition of a second copy of the scaffold protein gene cipA improved the activity on crystalline cellulose.

[0263] Table 10. Activity of cellulosomes purified from strains SC9 and SC10 on various substrates.

[0264] Activity (mU / mg) SC9 SC10 PASC 1423 1249 Avicel 27 30.5

[0265] In addition, strains SC8, SC9, and SC10 were grown on AECC, showing that strain SC10 grew faster ( Fig.12 , indicating that adding a second copy of the scaffolding protein gene cipA improves growth on lignocellulosic biomass).

[0266] The growth of C. acetobutylicum strain SC10 on AECC (using 55 g / L AECC in a pH-controlled fermentor) was also compared to the growth of C. cellulophagus under the same conditions. Fig.13 It was shown that Clostridium acetobutylicum strain SC10 was more efficient in utilizing AECC than Clostridium cellulophagus.

[0267] Finally, it was demonstrated that C. acetobutylicum strain SC10 was able to grow on crystalline cellulose, whereas C. acetobutylicum strains SC1 and SC9 were not able to grow on crystalline cellulose, with only very limited growth ( Fig.14 ).

[0268] List of strains used in the examples

[0269] The strains used in the examples are listed in Table 11 below.

[0270] Table 11. Description of the Clostridium acetobutylicum strains used in the Examples.

[0271]

[0272] Pthl-cipcel: The native cip-cel operon promoter was replaced with a thiolase promoter (pthl) in the native cip-cel operon. T1T2: A terminator and a second copy of orfxp were inserted between cel48A and cel5B. cel48A_E61Q: Glutamic acid at position 66 in Cel48A was replaced with glutamine. Pthl-cipcel(SAFA): The native cip-cel operon promoter was replaced with a thiolase promoter (pthl) in the SAFAcip-cel operon (containing the hybrid cel48SAFA gene instead of the native cel48A gene). Pthl-cel9: The promoter of the native cel9X gene was replaced with a thiolase promoter (pthl). ΔNrpE: The NrpE gene is deleted. Pthl-cel5Y: The native cel5Y gene promoter was replaced with a thiolase promoter (pthl). +xynB: an extra copy of the xynB gene was cloned downstream of the thiolase gene. +cipA-xynB: an extra copy of the cipA and xynB genes were cloned downstream of the thiolase gene.

[0273] List of plasmids used in the examples

[0274] The plasmids used in the examples and their sequences are listed below.

[0275] SEQ ID NO:1-pGRNA_Pthlcipcel:

[0276]

[0277]

[0278] SEQ ID NO:2-pGRNA_cel48A_T1T2

[0279]

[0280]

[0281] SEQ ID NO:3-pGRNA_cel48A_E61Q

[0282]

[0283]

[0284] SEQ ID NO:4-pGRNA_Pth1-cel9X

[0285]

[0286]

[0287] SEQ ID NO:5—PGRNA_ΔnrpE

[0288]

[0289] SEQ ID NO:6-pGRNA_Pthl_cel5Y

[0290]

[0291]

[0292] SEQ ID NO:9-pMTL_JH16-xynB

[0293]

[0294]

[0295]

[0296] SEQ ID NO:10-pMTL_JH16-cipA-xynB

[0297]

[0298]

[0299]

[0300]

[0301]

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Claims

1. A genetically modified Clostridium acetobutylicum capable of growing on lignocellulosic biomass, wherein the cip-cel operon is overexpressed, and each gene of the cip-cel operon is expressed at a higher level in the genetically modified Clostridium acetobutylicum than in the corresponding non-genetically modified Clostridium acetobutylicum.

2. The genetically modified Clostridium acetobutylicum according to claim 1, wherein: a) the native promoter of the cip-cel operon has been genetically modified, preferably: a1) the native promoter of the cip-cel operon has been replaced by a stronger promoter, a2) the native promoter of the cip-cel operon has been mutated, or b) an additional copy of the cip-cel operon has been inserted into the genome of C. acetobutylicum and is under the control of a stronger promoter than the native promoter of the cip-cel operon, The stronger promoter is preferably selected from the group consisting of Clostridium acetobutylicum thlA gene promoter, ptb gene, crt gene and gapC gene.

3. The genetically modified Clostridium acetobutylicum according to claim 2, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene.

4. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 3, wherein the cel48A gene encodes the native Cel48A protein of Clostridium acetobutylicum of the amino acid sequence WP_010964229.1, preferably the genetically modified Clostridium acetobutylicum comprises the native cel48A gene of the nucleic acid sequence corresponding to positions 1047700 to 1049880 of NC_003030.

1.

5. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 4, which is further genetically modified so that the cel9X gene is overexpressed, and the expression level of the cel9X gene in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

6. The genetically modified Clostridium acetobutylicum according to claim 5, wherein: a) The natural promoter of the cel9X gene is genetically modified, preferably: b1) The natural promoter of the cel9X gene has been replaced by a stronger promoter, b2) The native promoter of the cel9X gene has mutated, or b) an extra copy of the cel9X gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cel9X gene, The stronger promoter is preferably selected from the group consisting of Clostridium acetobutylicum thlA gene promoter, ptb gene, crt gene and gapC gene.

7. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 6, which is further genetically modified so that the cel5Y gene is overexpressed, and the expression level of the cel5Y gene in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

8. The genetically modified Clostridium acetobutylicum according to claim 7, wherein: a) The natural promoter of the cel5Y gene is genetically modified, preferably: a1) The native promoter of the cel5Y gene has been replaced by a stronger promoter, or a2) The natural promoter of the cel5Y gene has mutated, b) an extra copy of the cel5Y gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cel5Y gene, The stronger promoter is preferably selected from the group consisting of Clostridium acetobutylicum thlA gene promoter, ptb gene, crt gene and gapC gene.

9. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 8, which is further genetically modified so that the xynB gene is overexpressed, and the expression level of the xynB gene in the genetically modified Clostridium acetobutylicum is higher than its expression level in the corresponding non-genetically modified Clostridium acetobutylicum.

10. The genetically modified Clostridium acetobutylicum according to claim 9, wherein: a) an extra copy of the xynB gene has been inserted into the C. acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the xynB gene, b) the natural promoter of the xynB gene is genetically modified, preferably: b3) The native promoter of the xynB gene has been replaced by a stronger promoter, or b4) The natural promoter of the xynB gene has mutated, The stronger promoter is preferably selected from the group consisting of Clostridium acetobutylicum thlA gene promoter, ptb gene, crt gene and gapC gene.

11. A genetically modified Clostridium acetobutylicum according to any one of claims 1 to 10, which has been further genetically modified so that the cipA gene is further overexpressed relative to other genes of the cip-cel operon, this modification preferably resulting in an increase in the ratio of CipA scaffold protein produced to cellulosomal cellulase produced; preferably, an additional copy of the cipA gene has been inserted into the Clostridium acetobutylicum genome and is under the control of a stronger promoter than the native promoter of the cipA gene.

12. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 11, which has been further genetically modified by deleting a gene encoding an extracellular protease, preferably selected from the group consisting of nrpE, CA_C0746, CA_C0625 and CA_C2695.

13. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 12, selected from: a) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, b) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, c) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, d) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and an additional copy of the xynB gene is inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, e) a genetically modified Clostridium acetobutylicum, wherein the natural promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the natural promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted, f) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted, g) a genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, an extra copy of the xynB gene is inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted, and h) A genetically modified Clostridium acetobutylicum, wherein the native promoter of the cip-cel operon has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel9X gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, the native promoter of the cel5Y gene has been replaced by the promoter of the Clostridium acetobutylicum thlA gene, an additional copy of the xynB gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, an additional copy of the cipA gene has been inserted into the Clostridium acetobutylicum genome and is under the control of the promoter of the Clostridium acetobutylicum thlA gene, and the nrpE gene has been partially or completely deleted.

14. The genetically modified Clostridium acetobutylicum according to any one of claims 1 to 13, which has been further optimized to: a) producing ethanol by inactivating the ldhA gene, the thlA gene and the hydA gene, b) producing butanol by inactivating the ptb-buk operon, replacing the Clostridium acetobutylicum thlA gene with the Escherichia coli atoB gene, replacing the Clostridium acetobutylicum hbd gene with the Clostridium kluyveri hbd1 gene, and overexpressing the CA_C0764 gene to a level greater than that in a corresponding non-genetically modified Clostridium acetobutylicum; or c) Production of lactic acid by inactivation of the thlA gene and the hydA gene followed by elimination of the megaplasmid.

15. A method for producing a target commodity chemical from lignocellulosic biomass, comprising culturing the genetically modified Clostridium acetobutylicum of any one of claims 1 to 14 on a suitable culture medium comprising lignocellulosic biomass as a major carbon source, and recovering the target commodity chemical from the culture medium.

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