Patent Abstract:
An engineered bacterium for producing ethanol from one or more carbohydrates is disclosed. The bacterium can be made by (a) inactivating within a  Lactobacillus casei  bacterium one or more endogenous genes encoding a lactate dehydrogenase; or (b) introducing into a  Lactobacillus casei  bacterium one or more exogenous genes encoding a pyruvate decarboxylase and one or more exogenous genes encoding an alcohol dehydrogenase II; or (c) performing both steps (a) and (b). The resulting engineered bacterium produces significantly more ethanol than the wild-type  Lactobacillus casei  bacterium, and can be used in producing ethanol from a substrate such as biomass that includes carbohydrates.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/682,281 filed on Aug. 12, 2012, which is incorporated by reference herein in its entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    This invention was made with government support under DE-FC02-07ER64494 awarded by the US Department of Energy and 2011-67009-30043 awarded by the USDA/NIFA. The government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION 
       [0003]    This disclosure relates generally to a synthetic  Lactobacillus casei  bacterium engineered to produce increased amounts of ethanol, as compared to wild type  Lactobacillus casei , as well as to methods of making and using such a bacterium. 
       BACKGROUND OF THE INVENTION 
       [0004]    Microbial production of biofuels from lignocellulosic substrates is a component of the United States plan to reduce its dependency on fossil fuels. The microorganisms typically considered for the production of biofuels include  Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli , and  Clostridium  sp. However, all of these microorganisms suffer from one or more of the following deficiencies: relatively low tolerance to the environmental stresses likely to be encountered in fermentation (e.g., high levels of alcohols, acids, and/or osmolarity), complex physiology, poor availability of genetic tools, and limited ability to secrete enzymes. Accordingly, there is a need in the art for improved microorganisms for the production of biofuels such as ethanol from lignocellulosic substrates. 
       BRIEF SUMMARY OF THE INVENTION 
       [0005]    In a first aspect, this disclosure encompasses an engineered bacterium for producing ethanol from one or more carbohydrates. The engineered bacterium is made by (a) inactivating within a  Lactobacillus casei  bacterium one or more endogenous genes encoding a lactate dehydrogenase; or (b) introducing into a  Lactobacillus casei  bacterium one or more exogenous genes encoding a pyruvate decarboxylase and one or more exogenous genes encoding an alcohol dehydrogenase II. The engineered bacterium can also be made using a combination of both approaches. The resulting engineered bacterium produces significantly more ethanol than the wild type  Lactobacillus casei  bacterium. 
         [0006]    In certain embodiments, the  Lactobacillus casei  bacterium is made from  L. casei  strain 12A. 
         [0007]    In certain embodiments, the step of inactivating within a  Lactobacillus casei  bacterium one or more endogenous genes encoding a lactate dehydrogenase also includes inactivating within the  Lactobacillus casei  bacterium an endogenous gene encoding D-hydroxyisocaproate dehydrogenase. In certain embodiments, the engineered bacterium includes the gene deletion mutation Δ L-lactate dehydrogenase 1 (ΔL-ldh1), the gene deletion mutation Δ L-lactate dehydrogenase 2 (ΔL-ldh2), or both. In some such embodiments, the engineered bacterium further includes the gene deletion mutation Δ D-lactate dehydrogenase (ΔD-ldh) or Δ D-hydroxyisocaproate dehydrogenase (ΔD-hic). 
         [0008]    In certain embodiments, the exogenous gene encoding a pyruvate decarboxylase includes the gene of  Zymomonas mobilis  that encodes for pyruvate decarboxylase (Pdc), and the exogenous gene encoding an alcohol dehydrogenase II includes the gene of  Zymomonas mobilis  that encodes for dehydrogenase II (AdhII). Preferably, the exogenous genes are modified to utilize  L. casei  codon usage for highly expressed genes. 
         [0009]    In certain embodiments, the exogenous genes are introduced into the  L. casei  bacterium using an expression vector. A non-limiting example of an expression vector that could be used is pP pgm -PET. 
         [0010]    In certain embodiments, the exogenous genes are operably linked to a promoter. Preferably, the promoter is an  L. casei  promoter. An non-limiting example of a preferred  L. casei  promoter is the phosphoglycerate mutase (pgm) promoter (P pgm ). The  L. casei  promoter may also be a promoter that is highly expressed in the stationary phase. Non-limiting examples of such promoters include the  L. casei  GroEL promoter and the  L. casei  DnaK promoter. 
         [0011]    In a second aspect, the disclosure encompasses an engineered bacterium for producing ethanol from one or more carbohydrates. The engineered bacterium is a derivative of  L. casei  12A containing the deletion mutation ΔL-ldh1, an exogenous gene encoding a pyruvate decarboxylase, and an exogenous gene encoding an alcohol dehydrogenase II. The exogenous genes are operably linked to a native  L. casei  promoter, and the engineered bacterium produces significantly more ethanol than the wild-type  L. casei  bacterium. 
         [0012]    In certain embodiments, the engineered bacterium further includes the deletion mutation ΔL-ldh2. 
         [0013]    Non-limiting examples of native  L. casei  promoters that could be operably linked to the exogenous genes include the phosphoglycerate mutase promoter, the GroEL promoter, and the DnaK promoter. 
         [0014]    In certain embodiments, the exogenous genes are from  Zymomonas mobilis.    
         [0015]    In certain embodiments, the exogenous genes are included in a pP pgm -PET expression vector. In some such embodiments, the pgm promoter (P pgm ) in the pP pgm -PET expression vector may be substituted with a promoter that is highly expressed in the stationary phase. Non-limiting examples of such a promoter include a GroEL promoter or a DnaK promoter. 
         [0016]    In a third aspect, this disclosure encompasses a method of making ethanol. The method includes the step of culturing the engineered bacterium of any of the embodiments described above on a substrate comprising a carbohydrate, and collecting the ethanol produced by the bacterium. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1 . Growth (▪,) and glucose utilization (□,∘) by  Lactobacillus casei  12AΔ-ldh(pP PGM -PET) (squares) and 12AΔL-Idh1ΔL-Idh2ΔD-hic(pP PGM -PET) (circles) at 37° C. in modified chemical defined media (mCDM; Díaz-Muñiz and Steele, 2006) containing 10% glucose (w/v) with pH maintained at 6.0. See Díaz-Muñiz, I. and J. L. Steele, Antonie van Leeuwenhoek 90 (2006): 233-243. 
           [0018]      FIG. 2 . Growth, glucose consumption, and ethanol production by  Lactobacillus casei  12AΔLldh1(pP PGM -PET) (2A) and 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP PGM -PET) ( 2 B) at 37° C. in a chemically defined media containing 10% glucose with pH maintained at 6.0. 
           [0019]      FIG. 3 . Metabolism of pyruvate (PYR) in  Lactobacillus casei  12A and derivatives. The pyruvate related enzymes and pathways present in  L. casei  12A: L-lactate dehydrogenases (L-Ldh); D-lactate dehydrogenase (D-Ldh); D-Hydroxyisocaproate dehydrogenase (D-Hic); acetolactate synthase (Als); oxaloacetate decarboxylase (Oad); pyruvate kinase (Pyk); phophoenolpyruvate carboxikinase (Pck); pyruvate-formate lyase (Pfl); alcohol dehydrogenase (Adh). The enzymes and pathway from  Zymomonas mobilis  are shown as thick arrows: pyruvate decarboxylase (Pdc); alcohol dehydrogenase (Adh). Abbreviations: EMP, Embden-Meyerof-Parnas pathway; Glu, glucose; PEP, phosphoenolpyruvate. 
           [0020]      FIG. 4 . Schematic illustrating the gene replacement procedure developed for gene replacement in  L. casei  12A. Presence of the pheS* loci results in sensitivity to p-Cl-Phe. This phenotype (derivatives with pheS* form smaller colonies) allows for selection derivatives that have undergone recombination resulting in loss of the pheS* loci (derivatives without phe* form bigger colonies). 
           [0021]      FIG. 5 . “Production of ethanol” or PET cassette in pTRKH2 designed for  L casei  12A, called pP pgm -PET. Panel A, Construction of PET cassette in pTRKH2. PET cassette sequence was obtained from  Zymomonas mobilis . Codon usage of pdc and adhII were optimized specifically for  L. casei  12A using Java Codon Adaptation Tool (Jcat). Codon optimized cassette was synthesized then cloned into pTRKH2 for expression in  L casei  12A. Panel B, detail of gene organization in the PET cassette: Ppgm, native promoter from  L. casei  12A phosphoglycerate mutase; ribosomal binding site (RBS); pdc, pyruvate decarboxylase; adhII, alcohol dehydrogenase; Pin structure, native  L. casei  12A transcriptional terminator of kdgR transcriptional regulator protein. The cassette was flanked by PstI and BamHI restriction sites for cloning into pTRKH2. 
           [0022]      FIG. 6 . Growth curves of  L. casei  12A and 12A Δldh1 transformed with empty pTRKH2 (control) or pPgm-PET growth in chemically defined medium (CDM) for 48 hrs. Working cultures were prepared from frozen stocks by two sequential transfers in MRS broth (see J. C. de Man, M. Rogosa and M. Elisabeth Sharpe, Appl. Bact. 23. 130-135 (1960)) with incubations conducted statically at 37° C. for 24 hrs and 18 hrs, respectively. These cultures were then transferred to mCDM overnight and monitored every 6 hrs for OD600 (optical density at 600 nm). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. In General 
       [0023]    Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by any later-filed nonprovisional applications. 
         [0024]    As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. The terms “comprising”, “including”, and “having” can be used interchangeably. 
         [0025]    Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 
       II. The Invention 
       [0026]    We have developed a bioengineered biofuel-producing strain of  Lactobacillus casei . The following characteristics make  L. casei  an ideal biofuels fermentation organism: ability to use lignocellulosic-derived mono- and di-saccharides; resistance to environmental stresses likely to be encountered in industrial biofuels fermentations, including high levels of biofuels, acids, and/or osmolarity; relatively simple fermentative metabolism with almost complete separation of cellular processes for biosynthesis and energy metabolism; possibility to direct metabolic flux of both pentoses and hexoses to pyruvate (allowing for construction of derivatives producing second generation biofuels (i.e. isobutanol)); the availability of established platforms for introducing and expressing foreign DNA; availability of a deep portfolio of molecular-genetic data related to  L. casei  ecological adaptation, genomics, transcriptomics, lipidomics, and metabolomics; the ability to secrete and display proteins, hence potential for use in consolidated bioprocessing; and designation as a GRAS (Generally Regarded As Safe) species. 
         [0027]      L. casei  12A, a strain isolated from corn silage on the University of Wisconsin-Madison campus, was selected as the biofuels-producing parental strain, due to its alcohol resistance, carbohydrate utilization profile, and amenability to genetic manipulation. 
         [0028]    A two pronged approach has been employed to redirect metabolic flux in  L. casei  12A to ethanol. The first approach was to inactivate genes that encode enzymes which compete with the 12A pathway to ethanol. The second approach utilized the introduction of the genes from  Zymomonas mobilis  that encode pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (Adh2) activities (PET cassette). These genes were designed utilizing the  L. casei  codon usage for highly expressed genes with a constitutive  L. casei  promoter (phosphoglycerate mutase), synthesized, ligated with digested pTRKH2 to form pP PGM -PET), and introduced into 12A derivatives by electroporation. This two pronged approach has resulted in an  L. casei  12A derivative that produces ethanol as more than 80% of its metabolic end products. 
         [0029]    The constructed derivative of  L. casei  12A produces ethanol as more than 80% of its final metabolic end products from glucose, and the path to greater than 90% conversion is clear. This is by far the greatest conversion that has been reported with a lactobacilli, and will allow us to exploit the advantages of the use of lactobacilli as biocatalysts for the production of biofuels. These advantages are further delineated below. 
         [0030]    The specific features and advantages of the present invention will become apparent after a review of the following experimental examples. However, the invention is not limited to the specific embodiments disclosed herein. 
       III. Examples 
     Example A 
       [0031]    This example addresses (1) what level of carbohydrate  Lactobacillus casei  12A derivatives are capable of using; and (2) what level of ethanol production takes place at elevated glucose concentrations. 
         [0032]    In the first experiment, 48 small volume (2 ml) fermentations were conducted in GC vials containing our  L. casei  chemically defined media to examine glucose utilization and end product formation. In parallel, these fermentations were conducted in a 96 well plate reader to monitor growth. The experimental matrix was: 3 levels of glucose (2.5, 5.0, and 10% w/v), with and without the osmoprotectants present in ACSH (0.7 mM betaine, 0.7 mM choline chloride, and 0.2 mMDL-carnitine), with and without 2.5 μg/ml erythromycin (Ery) to select for the plasmid encoded PET cassette, and four different strains. The strains utilized were: (1) an  L. casei  12A derivative (12AΔL-ldh1) lacking L-lactate dehydrogenase 1 (L-ldh1), the primary fermentative lactate dehydrogenase, with pTRKH2 (empty vector control); (2) 12AΔL-ldh1 containing pPPGMPET, pTRKH2 with an insert containing the  L. casei  codon optimized  Zymomonas mobilis  genes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (Adh2) activities under the control of the  L. casei  phosphoglycerate mutase (pgm) promoter; (3) an  L. casei  12 A derivative (12AΔL-ldh1ΔL-ldh2ΔD-hic) lacking L-ldh1, L-ldh2, and D-hydroxyisocaproate dehydrogenase (D-Hic) containing pTRKH2; and (4) 12AΔL-ldh1ΔL-ldh2ΔD-hic containing pPPGM-PET. These fermentations were conducted at 37° C. for 96 h and the media had an initial pH of 6.0. 
         [0033]    Three of the strains (12AΔL-ldh1(pTRKH2), 12AΔL-ldh(pP PGM -PET) and 12AΔL-ldh1ΔLldh2ΔD-hic (pP PGM -PET) reached an OD600 of greater than 1.0 within 24 h and grew at indistinguishable rates regardless of the glucose concentration, the presence or absence of either osmoprotectants, or Ery. The other strain, 12AΔL-ldh1ΔL-Idh2ΔD-hic (pTRKH2) grew poorly, never reaching an OD600 of greater than 0.05, even after 96 h, regardless of media composition; this corresponds with previous experiments and was expected, as this strain lacks an efficient mechanism to regenerate NAD+ from pyruvate. 
         [0034]    The addition of osmoprotectants did not have a significant effect on growth of any of the strains under the conditions examined; however, the presence of the osmoprotectants did result in a reduction in lysis of strains producing ethanol in the presence of 2.5% glucose. No lysis was observed by the ethanol producing strains at the higher glucose concentrations, suggesting that the higher osmolarities induced genes that provide enhanced ethanol tolerance. The most significant finding from the growth experiments is that growth of  L. casei  12A derivatives is not affected by the glucose (osmolarity) concentrations up to 10%, rather these conditions seem to enhance cell viability in stationary phase of 12A derivatives producing ethanol. 
         [0035]    Metabolic end product accumulation in the small volume fermentations were determined by GLBRC Enabling Technologies (HPLC-RID), and the results for  L. casei  12AΔL-ldh(pP PGM PET) and 12AΔL-ldh1ΔL-ldh2ΔD-hic(pP PGM -PET) are presented in Table 1. All of the glucose was consumed in fermentations containing 2.5% (139 mM) and 5.0% (278 mM) glucose. In fermentations containing 10% (566 mM) glucose, glucose utilization ranged from 8.1 to 9.5% (459.1 to 536.4 mM). The ethanol formed in the 2.5% (139 mM) glucose fermentations ranged from 1.3 to 1.4% (219.6 to 247.6 mM), with % theoretical yields ranging from 79 to 89%. The ethanol formed in the 5.0% (278 mM) glucose fermentations ranged from 2.6 to 2.7% (438.0 to 466.0 mM), with % theoretical yields ranging from 79 to 84%. The ethanol formed in the 10% (566 mM) glucose fermentations ranged from 3.3 to 3.8% (563 to 651.5 mM), with % theoretical yields ranging from 50 to 58%. In fermentations containing 10% (556 mM) glucose, significant accumulation of pyruvate (73.2 to 92.4 mM) was observed, suggesting that pyruvate decarboxylase activity has become limiting. Under all the conditions examined,  L. casei  12AΔLldh1ΔL-ldh2ΔD-hic (pP PGM -PET) produced slightly more ethanol and slightly less lactate than  L. casei  12AΔl-ldh (pP PGM -PET). Possible reasons for incomplete glucose utilization in fermentations containing 10% glucose include changes in the pH of the media and increases in pressure due to conducting the fermentations in closed vials. To overcome these issues, fermentations that allow for pH control and CO 2  release have been conducted. 
         [0036]    Fermentations with 10% glucose with osmoprotectants and Ery have been conducted in our larger scale (500 ml) fermentation equipment that allows for pH control and CO 2  release with  L. casei  12AΔL-ldh (pP PGM -PET) and 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP PGM -PET) at 37° C., with pH maintained at 6.0. The growth and glucose utilization (enzymatic determination) results are presented in  FIG. 1 . Growth of the two strains are indistinguishable under these conditions; however, greater glucose utilization was observed by 12AΔL-ldh(pP PGM -PET). Metabolic end product accumulation in these fermentations was determined by GLBRC Enabling Technologies (HPLC-RID), and the results are presented in Example B. 
         [0037]    The 19 12A derivatives that were constructed via our two-step gene replacement method are presented in Table 2, clearly demonstrating the successful construction of a variety of 12A mutants. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Metabolic end products accumulated by  L. casei  12A ethanologens during growth 
               
               
                 in a chemically defined medium (initial pH 6.0) containing a different levels 
               
               
                 of glucose, with and without osmoprotectants at 37° C. for 96 hrs. 
               
             
          
           
               
                   
                 % 
               
             
          
           
               
                   
                 Osmo- 
                 Glucose (mM) 
                 Products (mM) 
                 Final 
                 Ethanol 
               
             
          
           
               
                 Strain 
                 protectant 
                 Int 
                 Con 
                 Rem 
                 EtOH 
                 Pyr 
                 Lac 
                 Ace 
                 pH 
                 (v/v)* 
               
               
                   
               
             
          
           
               
                 12A ΔL-Ldh1 
                 − 
                 142.0 
                 142.0 
                 BQL 
                 227.9 
                 BQL 
                 12.3 
                 6.6 
                 5.4 
                 1.3 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1 
                 − 
                 277.0 
                 277.0 
                 BQL 
                 438.0 
                 14.1 
                 26.6 
                 4.9 
                 4.7 
                 2.6 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1 
                 − 
                 499.7 
                 393.7 
                 106.0 
                 600.0 
                 73.2 
                 31.8 
                 5.3 
                 4.5 
                 3.5 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1 
                 + 
                 137.3 
                 137.3 
                 BQL 
                 219.6 
                 BQL 
                 11.8 
                 6.6 
                 6.2 
                 1.3 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1 
                 + 
                 278.1 
                 278.1 
                 BQL 
                 445.4 
                 3.3 
                 39.1 
                 3.2 
                 4.7 
                 2.6 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1 
                 + 
                 499.3 
                 469.6 
                 29.6 
                 563.0 
                 86.1 
                 40.5 
                 5.3 
                 4.4 
                 3.3 
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 − 
                 142.3 
                 142.3 
                 BQL 
                 247.6 
                 BQL 
                 7.7 
                 8.7 
                 7.4 
                 1.4 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 − 
                 282.6 
                 282.6 
                 BQL 
                 443.5 
                 18.3 
                 17.9 
                 9.1 
                 7.2 
                 2.6 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 − 
                 508.0 
                 401.1 
                 106.9 
                 625.0 
                 91.6 
                 22.3 
                 11.7 
                 6.6 
                 3.6 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 + 
                 139.5 
                 139.5 
                 BQL 
                 233.2 
                 BQL 
                 7.6 
                 8.0 
                 6.1 
                 1.4 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 + 
                 280.9 
                 280.9 
                 BQL 
                 466.0 
                 18.1 
                 15.2 
                 8.3 
                 6.7 
                 2.7 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 12A ΔL-Ldh1/ 
                 + 
                 507.9 
                 408.9 
                 99.0 
                 651.5 
                 92.4 
                 26.0 
                 12.4 
                 6.8 
                 3.8 
               
               
                 ΔL-Ldh2/ΔD-Hic 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (pP PGM -PET) 
               
               
                   
               
               
                 Abbreviations: BQL = Below Quantitative Level. Abbr: Int—initial, Con—consumed, Rem—remaining, EtOH—ethanol, Pyr—pyruvate, Lac—Lactate, Ace—acetate. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   Lactobacillus casei  12A derivatives constructed in the past 10 months in the Steele 
               
               
                 laboratory via gene replacement. 
               
             
          
           
               
                 Single knockouts 
                 Double knockouts 
                 Triple knockouts 
                 Quadruple knockouts 
               
               
                   
               
               
                 ΔL-ldh1* 
                 ΔL-ldh1/ΔL-ldh2* 
                 ΔL-ldh1/ΔL-ldh2/ΔL-ldh3 
                 ΔoadA/Δpck/Δpyc/Δfum 
               
               
                 ΔL-ldh2* 
                 ΔL-ldh1/ΔL-ldh3 
                 ΔL-ldh1/ΔL-ldh2/ΔL-ldh4 
                 ΔoadA/Δpck/Δpyc/Δaspal 
               
               
                 ΔL-ldh3 
                 ΔL-ldh1/ΔL-ldh4 
                 ΔL-ldh1/ΔLldh2/ΔD-ldh 
               
               
                 ΔL-ldh4 
                 ΔL-ldh1/ΔD-ldh 
                 ΔL-ldh1/ΔL-ldh2/ΔD-hic* 
               
               
                 ΔD-ldh 
                 ΔL-ldh1/ΔD-hic 
                 ΔoadA/Δpck/Δpyc 
               
               
                 ΔD-hic 
                 ΔL-ldh1/Δpck 
               
               
                 Δals 
                 ΔoadA/Δpck 
               
               
                 Δald 
                 ΔoadA/Δpyc 
               
               
                 Δaldrc 
                 Δpyc/Δpck 
               
               
                 ΔoadA 
               
               
                 Δpyc 
               
               
                 Δpck 
               
               
                 Δaspal 
               
               
                   
               
               
                 Abbreviations: 
               
               
                 L-ldh—L-lactate dehydrogenase, 
               
               
                 D-ldh—D-lactate dehydrogenase, 
               
               
                 D-hic—D-hydroxyisocaproate dehydrogenase, 
               
               
                 als—acetolactate synthase, 
               
               
                 ald—alpha acetolactate decarboxylase, 
               
               
                 a/drc—acetoin/diacetyl reductase, 
               
               
                 oad—oxaloacetate decarboxylase, 
               
               
                 pyc—pyruvate carboxylase, 
               
               
                 pck—phosphoenolpyruvate carboxikinase. 
               
               
                 aspal—aspartate-ammonia lyase, 
               
               
                 fum—fumarase. 
               
               
                 Asterisk - derivatives transformed with pP PGM -PET are available. 
               
             
          
         
       
     
       Example B 
       [0038]    This example shows the analysis of the data we obtained from the fermentations with 10% glucose with osmoprotectants and Ery that were conducted in our larger scale (500 ml) fermentation equipment with  Lactobacillus casei  12AΔL-ldh (pP PGM -PET) and 12AΔL-ldh1ΔLldh2ΔD-hic (pP PGM -PET) at 37° C., with pH maintained at 6.0. We could only accommodate three fermentation vessels at a time. Therefore, only the 12AΔL-ldh (pP PGM -PET) fermentation was conducted in duplicate. 
         [0039]    The growth, glucose utilization, and ethanol production shown by these strains are presented in  FIGS. 2A  ( L. casei  12AΔL-ldh (pP PGM -PET)) and  2 B ( L. casei  12AΔL-ldh1ΔLldh2ΔD-hic (pP PGM -PET)). The growth of the two strains under these conditions was indistinguishable. However 12AΔL-ldh (pP PGM -PET) utilized a greater quantity of glucose and produced more ethanol than 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP PGM -PET). The glucose utilization and ethanol formation obtained with 12AΔL-ldh (pP PGM -PET) in the larger fermentation vessels was significantly greater than that obtained in the small volume fermentations described in Example A. The mostly likely reason for this difference is that the larger vessels allow for pH control. 
         [0040]    The metabolic end products formed and glucose utilized as a function of time for these fermentations is presented in Tables 3 and 4. 12AΔL-ldh1 (pP PGM -PET) will be the focus of this discussion, due to its higher productivity. This 12A derivative utilized 504.5 mM glucose (9.1%) glucose in 96 h and produced 934.7 mM of “pyruvate-derived” metabolic end products, which is 87.4% of the theoretical yield from glucose. Ethanol was produced at a level of 771.3 mM (4.5%), which was 82.5% of the metabolic end-products. 
         [0041]    The second most abundant metabolic end product was pyruvate, which was present at 110.1 mM after 96 h. Pyruvate accumulation began at approximately 21 h, at the same time, ethanol as a percentage of the total metabolic end products began to decrease (% ethanol in total), suggesting that pyruvate decarboxylase activity becomes limiting at that time. This corresponds to the entry of this organism into stationary phase, suggesting that the  L. casei  phosphoglycerate mutase (pgm) promoter used to drive expression of the PET cassette is poorly expressed in stationary phase. It is highly likely that pyruvate accumulation can be overcome by utilizing a  L. casei  promoter highly expressed in stationary phase. If the pyruvate, which had accumulated after 96 h in the 12AΔL-ldh1(pPPGM-PET) fermentation, had been converted to ethanol, a total of 881.4 mM (5.14%) ethanol would have been produced. Additionally, the rate of glucose utilization would have been even higher, as pyruvate accumulation is known to inhibit glycolysis. 
         [0042]    It is difficult to directly compare our results to what is known concerning other biocatalysts, due to differences in media and fermentation equipment utilized. However, the results obtained in these  L. casei  12AΔL-ldh1 (pP PGM -PET) fermentations are most similar to the  Escherichia coli  GLBRCE1 synthetic hydrolysate fermentations reported by Schwalbach et al. (2012, AEM 78:3442) in  E. coli . GLBRCE1 converted 338 mM glucose into 477 mM ethanol, an ethanol yield of 70.5% of the theoretical maximum.  L. casei  12AΔL-ldh1 (pP PGM -PET) converted 504.5 mM glucose into 771.3 mM ethanol, an ethanol yield of 76.4% of the theoretical maximum. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Metabolic end products formed and glucose consumption by  Lactobacillus casei   
               
               
                 12A ΔL-Ldh1 (pP PGM -PET) at 37° C. in a chemically defined 
               
               
                 media containing 10% glucose with pH maintained at 6.0. 
               
             
          
           
               
                   
                 % 
                 % 
                   
               
             
          
           
               
                 Time 
                 Glucose (mM) 
                 Products (mM) 
                 % 
                 Ethanol 
                 Ethanol 
                 Ethanol:Lactate 
               
             
          
           
               
                 (hr) 
                 Rem 
                 Con 
                 Total 
                 EtOH 
                 Pyr 
                 Lac 
                 Ace 
                 yield 
                 in total 
                 (v/v) 
                 ratio (mM:mM) 
               
               
                   
               
             
          
           
               
                 0 
                 534.9 
                 BQL 
                 12.5 
                 11.1 
                 0.0 
                 0.1 
                 1.2 
                 1.2 
                 89.2 
                 0.1 
                 85 
               
               
                 1 
                 540.6 
                 BQL 
                 22.7 
                 12.0 
                 0.0 
                 5.4 
                 5.3 
                 2.1 
                 52.7 
                 0.1 
                 2 
               
               
                 2 
                 545.2 
                 BQL 
                 24.0 
                 12.8 
                 0.0 
                 5.5 
                 5.7 
                 2.2 
                 53.4 
                 0.1 
                 2 
               
               
                 3 
                 544.8 
                 BQL 
                 19.2 
                 12.8 
                 0.0 
                 2.5 
                 3.8 
                 1.8 
                 67.0 
                 0.1 
                 5 
               
               
                 4 
                 536.5 
                 BQL 
                 21.5 
                 15.0 
                 0.0 
                 2.3 
                 4.2 
                 2.0 
                 70.0 
                 0.1 
                 7 
               
               
                 5 
                 531.4 
                 BQL 
                 33.5 
                 27.1 
                 0.0 
                 1.3 
                 5.0 
                 3.1 
                 81.0 
                 0.2 
                 20 
               
               
                 6 
                 544.4 
                 BQL 
                 28.2 
                 21.7 
                 0.0 
                 0.9 
                 5.6 
                 2.6 
                 77.0 
                 0.1 
                 25 
               
               
                 7 
                 542.2 
                 BQL 
                 36.8 
                 29.4 
                 0.0 
                 1.8 
                 5.7 
                 3.4 
                 79.8 
                 0.2 
                 17 
               
               
                 8 
                 551.2 
                 BQL 
                 42.3 
                 34.4 
                 0.0 
                 2.1 
                 5.8 
                 4.0 
                 81.3 
                 0.2 
                 16 
               
               
                 9 
                 539.9 
                 BQL 
                 51.2 
                 42.8 
                 0.0 
                 2.7 
                 5.8 
                 4.8 
                 83.5 
                 0.2 
                 16 
               
               
                 10 
                 535.1 
                 BQL 
                 67.0 
                 58.1 
                 0.0 
                 3.3 
                 5.6 
                 6.3 
                 86.7 
                 0.3 
                 18 
               
               
                 11 
                 513.9 
                 21.0 
                 74.9 
                 66.1 
                 0.0 
                 3.6 
                 5.2 
                 7.0 
                 88.2 
                 0.4 
                 18 
               
               
                 12 
                 521.1 
                 13.8 
                 90.6 
                 82.5 
                 0.0 
                 2.9 
                 5.2 
                 8.5 
                 91.0 
                 0.5 
                 28 
               
               
                 13 
                 512.8 
                 22.1 
                 105.1 
                 96.8 
                 0.0 
                 3.4 
                 4.9 
                 9.8 
                 92.1 
                 0.6 
                 29 
               
               
                 14 
                 506.3 
                 28.7 
                 124.5 
                 116.1 
                 0.0 
                 3.9 
                 4.4 
                 11.6 
                 93.3 
                 0.7 
                 29 
               
               
                 15 
                 490.8 
                 44.1 
                 140.0 
                 131.7 
                 0.0 
                 4.4 
                 4.0 
                 13.1 
                 94.0 
                 0.8 
                 30 
               
               
                 16 
                 481.5 
                 53.5 
                 158.7 
                 150.1 
                 0.0 
                 4.9 
                 3.7 
                 14.8 
                 94.6 
                 0.9 
                 31 
               
               
                 17 
                 463.4 
                 71.5 
                 175.4 
                 166.6 
                 0.0 
                 5.4 
                 3.4 
                 16.4 
                 95.0 
                 1.0 
                 31 
               
               
                 18 
                 439.5 
                 95.4 
                 196.7 
                 187.6 
                 0.0 
                 5.9 
                 3.2 
                 18.4 
                 95.4 
                 1.1 
                 32 
               
               
                 19 
                 442.9 
                 92.0 
                 223.0 
                 213.2 
                 0.0 
                 6.7 
                 3.1 
                 20.8 
                 95.6 
                 1.2 
                 32 
               
               
                 20 
                 419.4 
                 115.5 
                 235.4 
                 225.5 
                 0.0 
                 7.0 
                 2.8 
                 22.0 
                 95.8 
                 1.3 
                 32 
               
               
                 21 
                 414.8 
                 120.1 
                 258.5 
                 247.6 
                 0.4 
                 7.8 
                 2.7 
                 24.2 
                 95.8 
                 1.4 
                 32 
               
               
                 22 
                 402.2 
                 132.7 
                 275.1 
                 263.0 
                 1.1 
                 8.4 
                 2.7 
                 25.7 
                 95.6 
                 1.5 
                 31 
               
               
                 23 
                 389.1 
                 145.8 
                 292.6 
                 278.7 
                 2.1 
                 9.2 
                 2.5 
                 27.3 
                 95.3 
                 1.6 
                 30 
               
               
                 24 
                 412.0 
                 122.9 
                 297.6 
                 281.5 
                 3.4 
                 9.8 
                 2.8 
                 27.8 
                 94.6 
                 1.6 
                 29 
               
               
                 25 
                 375.9 
                 159.0 
                 336.1 
                 318.4 
                 4.4 
                 10.8 
                 2.5 
                 31.4 
                 94.7 
                 1.9 
                 29 
               
               
                 26 
                 357.3 
                 177.6 
                 353.2 
                 332.4 
                 5.7 
                 11.8 
                 3.3 
                 33.0 
                 94.1 
                 1.9 
                 28 
               
               
                 27 
                 343.8 
                 191.1 
                 363.0 
                 339.7 
                 7.8 
                 12.4 
                 3.1 
                 33.9 
                 93.6 
                 2.0 
                 27 
               
               
                 28 
                 339.7 
                 195.2 
                 387.7 
                 362.0 
                 9.4 
                 13.3 
                 2.9 
                 36.2 
                 93.4 
                 2.1 
                 27 
               
               
                 29 
                 336.6 
                 198.3 
                 411.0 
                 382.9 
                 10.5 
                 14.3 
                 3.2 
                 38.4 
                 93.2 
                 2.2 
                 27 
               
               
                 30 
                 318.4 
                 216.5 
                 411.7 
                 383.1 
                 11.1 
                 14.7 
                 2.9 
                 38.5 
                 93.0 
                 2.2 
                 26 
               
               
                 32 
                 292.4 
                 242.5 
                 451.8 
                 421.5 
                 12.9 
                 15.1 
                 2.3 
                 42.2 
                 93.3 
                 2.5 
                 28 
               
               
                 34 
                 289.7 
                 245.2 
                 481.8 
                 445.1 
                 16.3 
                 17.4 
                 2.9 
                 45.0 
                 92.4 
                 2.6 
                 26 
               
               
                 44 
                 221.6 
                 313.3 
                 588.7 
                 533.1 
                 29.3 
                 22.8 
                 3.4 
                 55.0 
                 90.6 
                 3.1 
                 23 
               
               
                 50 
                 187.2 
                 347.7 
                 656.5 
                 584.7 
                 41.6 
                 25.7 
                 4.4 
                 61.4 
                 89.1 
                 3.4 
                 23 
               
               
                 58 
                 151.4 
                 383.5 
                 714.0 
                 623.4 
                 56.3 
                 28.5 
                 5.8 
                 66.7 
                 87.3 
                 3.6 
                 22 
               
               
                 66 
                 118.6 
                 416.3 
                 768.6 
                 664.1 
                 65.3 
                 31.5 
                 7.7 
                 71.8 
                 86.4 
                 3.9 
                 21 
               
               
                 70 
                 99.9 
                 435.0 
                 813.2 
                 691.8 
                 78.8 
                 33.4 
                 9.2 
                 76.0 
                 85.1 
                 4.0 
                 21 
               
               
                 74 
                 92.2 
                 442.7 
                 827.3 
                 702.8 
                 81.3 
                 33.8 
                 9.4 
                 77.3 
                 85.0 
                 4.1 
                 21 
               
               
                 82 
                 62.4 
                 472.5 
                 873.8 
                 744.0 
                 81.1 
                 36.8 
                 11.9 
                 81.7 
                 85.1 
                 4.3 
                 20 
               
               
                 90 
                 44.1 
                 490.8 
                 913.8 
                 764.4 
                 97.8 
                 38.3 
                 13.2 
                 85.4 
                 83.7 
                 4.5 
                 20 
               
               
                 96 
                 30.4 
                 504.5 
                 934.7 
                 771.3 
                 110.1 
                 39.2 
                 14.1 
                 87.4 
                 82.5 
                 4.5 
                 20 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Metabolic end products formed and glucose consumption by  Lactobacillus casei   
               
               
                 12A ΔL-Ldh1/ΔL-Ldh2/ΔD-Hic (pP PGM -PET) at 37° C. in a 
               
               
                 chemically defined media containing 10% glucose with pH maintained at 6.0. 
               
             
          
           
               
                   
                 % 
                   
                   
               
               
                   
                 Ethanol 
                 % 
                   
               
             
          
           
               
                 Time 
                 Glucose (mM) 
                 Products (mM) 
                 % 
                 in total 
                 Ethanol 
                 Ethanol:Lactate 
               
             
          
           
               
                 (hr) 
                 Rem 
                 Con 
                 Total 
                 EtOH 
                 Pyr 
                 Lac 
                 Ace 
                 yield 
                 product 
                 (v/v) 
                 ratio (mM:mM) 
               
               
                   
               
             
          
           
               
                 0 
                 575.3 
                 BQL 
                 13.3 
                 13.3 
                 0.0 
                 0.0 
                 0.0 
                 1.2 
                 100.0 
                 0.1 
                 — 
               
               
                 1 
                 557.1 
                 18.3 
                 12.9 
                 12.9 
                 0.0 
                 0.0 
                 0.0 
                 1.1 
                 100.0 
                 0.1 
                 — 
               
               
                 2 
                 553.1 
                 22.3 
                 12.7 
                 12.5 
                 0.0 
                 0.0 
                 0.2 
                 1.1 
                 98.1 
                 0.1 
                 — 
               
               
                 3 
                 566.0 
                 9.3 
                 15.2 
                 14.5 
                 0.0 
                 0.0 
                 0.6 
                 1.3 
                 96.0 
                 0.1 
                 — 
               
               
                 4 
                 541.8 
                 33.6 
                 16.7 
                 15.2 
                 0.0 
                 0.0 
                 1.5 
                 1.5 
                 90.9 
                 0.1 
                 — 
               
               
                 5 
                 548.8 
                 26.5 
                 21.7 
                 18.7 
                 0.0 
                 0.0 
                 3.0 
                 1.9 
                 86.2 
                 0.1 
                 — 
               
               
                 6 
                 543.2 
                 32.1 
                 25.8 
                 21.0 
                 0.0 
                 0.0 
                 4.9 
                 2.2 
                 81.2 
                 0.1 
                 — 
               
               
                 7 
                 551.2 
                 24.1 
                 31.6 
                 25.5 
                 0.0 
                 0.0 
                 6.1 
                 2.7 
                 80.6 
                 0.1 
                 — 
               
               
                 8 
                 554.9 
                 20.4 
                 36.8 
                 30.6 
                 0.0 
                 0.0 
                 6.2 
                 3.2 
                 83.1 
                 0.2 
                 — 
               
               
                 9 
                 551.3 
                 24.0 
                 45.6 
                 38.5 
                 0.0 
                 1.1 
                 6.0 
                 4.0 
                 84.4 
                 0.2 
                 36 
               
               
                 10 
                 532.7 
                 42.6 
                 55.5 
                 48.7 
                 0.0 
                 0.9 
                 5.9 
                 4.8 
                 87.6 
                 0.3 
                 53 
               
               
                 11 
                 532.3 
                 43.0 
                 64.5 
                 57.7 
                 0.0 
                 1.1 
                 5.8 
                 5.6 
                 89.4 
                 0.3 
                 54 
               
               
                 12 
                 544.0 
                 31.3 
                 76.6 
                 70.6 
                 0.0 
                 0.0 
                 6.1 
                 6.7 
                 92.1 
                 0.4 
                 — 
               
               
                 13 
                 536.6 
                 38.7 
                 87.7 
                 82.1 
                 0.0 
                 0.0 
                 5.6 
                 7.6 
                 93.6 
                 0.5 
                 — 
               
               
                 14 
                 519.6 
                 55.8 
                 101.5 
                 93.7 
                 0.0 
                 2.6 
                 5.2 
                 8.8 
                 92.3 
                 0.5 
                 36 
               
               
                 15 
                 510.2 
                 65.1 
                 111.9 
                 107.0 
                 0.0 
                 0.0 
                 4.9 
                 9.7 
                 95.7 
                 0.6 
                 — 
               
               
                 16 
                 513.9 
                 61.4 
                 134.2 
                 125.5 
                 0.0 
                 3.8 
                 4.8 
                 11.7 
                 93.6 
                 0.7 
                 33 
               
               
                 17 
                 474.2 
                 101.1 
                 134.7 
                 130.7 
                 0.0 
                 0.0 
                 3.9 
                 11.7 
                 97.1 
                 0.8 
                 — 
               
               
                 18 
                 485.3 
                 90.0 
                 167.7 
                 158.3 
                 0.0 
                 5.5 
                 3.9 
                 14.6 
                 94.4 
                 0.9 
                 29 
               
               
                 19 
                 463.7 
                 111.6 
                 179.9 
                 170.4 
                 0.0 
                 5.9 
                 3.5 
                 15.6 
                 94.8 
                 1.0 
                 29 
               
               
                 20 
                 462.1 
                 113.2 
                 199.4 
                 189.4 
                 0.0 
                 6.8 
                 3.2 
                 17.3 
                 95.0 
                 1.1 
                 28 
               
               
                 21 
                 459.0 
                 116.3 
                 218.4 
                 207.8 
                 0.0 
                 7.6 
                 3.0 
                 19.0 
                 95.1 
                 1.2 
                 27 
               
               
                 22 
                 451.9 
                 123.5 
                 236.9 
                 224.9 
                 0.7 
                 8.6 
                 2.8 
                 20.6 
                 94.9 
                 1.3 
                 26 
               
               
                 23 
                 426.3 
                 149.0 
                 238.0 
                 235.8 
                 0.0 
                 0.0 
                 2.3 
                 20.7 
                 99.0 
                 1.4 
                 — 
               
               
                 24 
                 380.6 
                 194.7 
                 314.8 
                 299.9 
                 3.5 
                 9.4 
                 1.9 
                 27.4 
                 95.3 
                 1.8 
                 32 
               
               
                 25 
                 426.0 
                 149.3 
                 295.3 
                 279.1 
                 3.6 
                 10.4 
                 2.2 
                 25.7 
                 94.5 
                 1.6 
                 27 
               
               
                 26 
                 384.5 
                 190.8 
                 297.1 
                 283.3 
                 2.5 
                 9.6 
                 1.7 
                 25.8 
                 95.3 
                 1.7 
                 29 
               
               
                 27 
                 365.0 
                 210.3 
                 301.5 
                 289.6 
                 0.5 
                 9.6 
                 1.8 
                 26.2 
                 96.1 
                 1.7 
                 30 
               
               
                 28 
                 371.3 
                 204.1 
                 331.6 
                 317.2 
                 1.7 
                 11.0 
                 1.6 
                 28.8 
                 95.7 
                 1.9 
                 29 
               
               
                 29 
                 360.5 
                 214.8 
                 339.2 
                 320.3 
                 5.6 
                 10.9 
                 2.3 
                 29.5 
                 94.4 
                 1.9 
                 29 
               
               
                 30 
                 332.9 
                 242.4 
                 341.2 
                 326.4 
                 2.9 
                 10.9 
                 1.0 
                 29.7 
                 95.7 
                 1.9 
                 30 
               
               
                 32 
                 333.5 
                 241.8 
                 395.1 
                 375.6 
                 5.8 
                 12.5 
                 1.2 
                 34.3 
                 95.1 
                 2.2 
                 30 
               
               
                 34 
                 296.7 
                 278.6 
                 381.7 
                 363.9 
                 5.3 
                 12.1 
                 0.4 
                 33.2 
                 95.3 
                 2.1 
                 30 
               
               
                 44 
                 272.5 
                 302.8 
                 520.6 
                 480.5 
                 22.4 
                 14.4 
                 3.2 
                 45.2 
                 92.3 
                 2.8 
                 33 
               
               
                 50 
                 245.1 
                 330.2 
                 578.6 
                 527.3 
                 30.5 
                 16.6 
                 4.2 
                 50.3 
                 91.1 
                 3.1 
                 32 
               
               
                 58 
                 216.0 
                 359.4 
                 627.0 
                 564.1 
                 39.4 
                 17.6 
                 5.8 
                 54.5 
                 90.0 
                 3.3 
                 32 
               
               
                 66 
                 194.1 
                 381.3 
                 681.0 
                 606.2 
                 49.2 
                 18.3 
                 7.2 
                 59.2 
                 89.0 
                 3.5 
                 33 
               
               
                 70 
                 175.2 
                 400.1 
                 700.5 
                 618.2 
                 54.8 
                 19.5 
                 8.0 
                 60.9 
                 88.3 
                 3.6 
                 32 
               
               
                 74 
                 169.5 
                 405.8 
                 706.8 
                 622.6 
                 56.7 
                 19.2 
                 8.3 
                 61.4 
                 88.1 
                 3.6 
                 32 
               
               
                 82 
                 149.8 
                 425.5 
                 712.2 
                 639.6 
                 63.1 
                 0.0 
                 9.5 
                 61.9 
                 89.8 
                 3.7 
                 — 
               
               
                 90 
                 141.0 
                 434.3 
                 752.9 
                 672.0 
                 70.1 
                 0.0 
                 10.8 
                 65.4 
                 89.3 
                 3.9 
                 — 
               
               
                 96 
                 126.6 
                 448.7 
                 774.2 
                 663.3 
                 79.0 
                 21.1 
                 10.7 
                 67.3 
                 85.7 
                 3.9 
                 31 
               
               
                   
               
               
                 Abbreviations in Tables: Rem, Remaining; Con, consumed; EtOH, ethanol; Pyr, pyruvate; Lac, lactate; Ace, acetate. 
               
               
                 % yield = (mM Total product/(2 × mM initial Glucose)) × 100 
               
               
                 % Ethanol = (mmol/L ethanol × 46.068 g/mol)/(1000 mg/g) × (1000 ml/L/100 ml) × (0.789 g/ml). 
               
             
          
         
       
     
       Example C 
     Screening Strains of  L. casei  for Biofuels Relevant Phenotypes and Genes 
       [0043]    Our laboratory has a culture collection contains approximately 60 strains of  L. casei  isolated from green plant material (i.e. corn silage), cheese, wine, and humans. The eleven strains with genome sequences were screened for the ability to utilize 60 different carbohydrates, including numerous carbohydrates present in lignocellulosic feed stocks. Individual strains were able to grow on between 17 and 26 different substrates. The strains isolated from corn silage (12A and 32G) grew on the greatest number of substrates. Nine gene clusters potentially involved in cellobiose utilization and one gene cluster involved in xylose utilization were identified. 
         [0044]    The eleven strains with genomic information were also screened for alcohol tolerance (ethanol, 1-propanol, 1-butanol, and 2-methyl-1-butanol), growth in AFEX-pretreated corn stover hydrolysate (ACSH), and transformation (electroporation) efficiency.  L. casei  12A exhibited the greatest tolerance to the biofuels examined. For example, when grown in the presence of 10% ethanol, it reached a final cell density 40% of that it attained in the absence of ethanol. Of the 11 strains examined for growth in corn stover hydrolysate, 3 of these strains (ATCC 334, 21-1, and 12A) grew significantly better, reaching a final optical density at 600 nm of approximately 2.0 within 28 h. Five  L. casei  strains were examined for transformation efficiency with pTRKH2 (O&#39;Sullivan and Klaenhammer 1993).  L. casei  12A exhibited a frequency (approximately 5×10 5  transformants per ug of pTRKH2) at least 50-fold higher than that observed with any of the other strains examined. Based upon the results from these analyses,  L. casei  12A was selected as the biofuel producing parental strain. 
         [0045]    Completing the  L. casei  12A genome. For further information regarding the  L. casei  12A genome, see Broadbent, et al.,  BMC Genomics  2012, 13:533, which is incorporated by reference herein. To enhance the depth of genomic sequence coverage of 12A, genomic DNA was prepared and submitted to the Joint Genome Institute (JGI) for genome sequencing. A draft genome of  L. casei  12A with approximately 500× coverage assembled into 397 scaffolds was received from JGI. This genome assembly was subsequently merged with the previous 23×454-generated paired end genome assembly in collaboration with personnel from DuPont Inc. (Madison, Wis.), yielding a genome assembly with 19 ordered contigs. We have generated PCR amplicons across all 19 gaps, and have sequenced  10  of these amplicons. 
         [0046]      L. casei  Metabolic Models. 
         [0047]    We have developed a genome-scale metabolic model for  L. casei  ATCC334 (the neotype strain) and 12A using the ModelSEED database and the genome annotation from RAST. We have modified the draft  L. casei  12A model from ModelSEED using the following processes: 1) thermodynamically infeasible cycles were removed, 2) elementally imbalanced metabolic reactions were corrected; and 3) model predictions for amino acid requirements were compared against experimental growth phenotypes determined in a lactobacilli chemically defined medium (CDM) described by Christensen and Steele (J. Bacteriol. 185 (2003): 3297-3306). Inconsistencies were corrected by the addition or deletion of some reactions. 
         [0048]    Redirecting Metabolic Flux in  L. casei  12A to Ethanol. 
         [0049]    The development of a method to inactivate genes in  L. casei  was a requirement for the construction of a  L. casei  strain capable of converting lignocellulosic biomass to ethanol. An efficient gene replacement method based on the introduction of pCJK47-based constructs (Kristich et al. 2007) via a 12A optimized electroporation protocol was developed. 
         [0050]    A two pronged approach was employed to redirect metabolic flux in  L. casei  12A to ethanol. The first approach is to inactivate genes that encode enzymes which compete with the 12A pathway to ethanol, which has acetyl-CoA as an intermediate. There are a large number of genes that encode enzymes potentially involved in anaerobic pyruvate metabolism in  L. casei . We have inactivated 9 of these genes: pyruvate-formate lyase (Pfl), the four L-lactate dehydrogenases (L-ldh1, Lldh2, L-ldh3, and L-ldh4), D-lactate dehydrogenase (D-ldh), D-hydroxyisocaproate dehydrogenase (DHic), acetolactate synthase (Als), and oxaloacetate decarboxylase (OadA). Additionally, 5 derivatives lacking two or three of the dehydrogenases have been constructed. Characterization of the end product distribution these mutants is presented in Table 5. The highest level of metabolic redirection to ethanol achieved to date using this approach, is 21%, achieved with the 12A ΔL-ldh1ΔL-ldh2ΔD-hic derivative. It is interesting to note that this derivative also accumulates pyruvate. 
         [0051]    The second approach utilized to direct metabolic flux in 12A towards ethanol was the introduction of the genes from  Zymomonas mobilis  that encode pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (Adh2) activities (PET cassette). These genes were designed utilizing the  L. casei  codon usage for highly expressed genes with a constitutive  L. casei  promoter (phosphoglycerate mutase), synthesized by GeneArt, ligated with digested pTRKH2 (pPGM-PET), and introduced into 12A derivatives by electroporation. Characterization of the end product distribution of two of these derivatives has been completed and is presented in Table 5. The highest level of metabolic redirection to ethanol achieved to date using this approach is 85.3%, achieved with the 12A ΔL-ldh1ΔL-ldh2 (pP pgm -PET) derivative. It is interesting to note that 12A derivatives with pP pgm -PET grow more rapidly than their corresponding strains, suggesting that ethanol is less inhibitory to 12A derivatives than lactate. 
         [0052]    These results suggest that the two pronged approach is effective for redirecting 12A metabolic flux to ethanol. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Growth, substrate consumption, and metabolic end products formed by  Lactobacillus casei   
               
               
                 12A and derivatives during growth in a chemically defined media at 37° C. for 48 hrs. 
               
             
          
           
               
                   
                 Concentration (mM) 
                   
               
             
          
           
               
                   
                 Growth 
                 Substrate 
                 Metabolic End Products 
                   
                 EtOH/ 
               
             
          
           
               
                   
                 Max 
                 T           
                 Utilization a   
                 (% of total) b   
                 Yield 
                 Lac 
               
             
          
           
               
                 Derivative 
                 OD           
                 (h) 
                 Glc 
                 Cit 
                 Total 
                 L-lac 
                 D-lac 
                 EtOH 
                 Ace 
                 Pyr 
                 (%) c   
                 ratio d   
               
               
                   
               
             
          
           
               
                 12A 
                 1.05 
                 8.1 
                 51.5 
                 0.6 
                 112.2 
                 105.4, 
                 3.3 
                 1.4, 
                 2.1, 
                 BQL 
                 108 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (94) 
                 (3) 
                 (1) 
                 (2) 
                   
                   
                   
               
               
                 12A ΔL-ldh1 
                 1.28 
                 7.0 
                 52.6 
                 11.5 
                 87.0 
                 42.3, 
                 28.2, 
                 16.5, 
                 BQL 
                 BQL 
                 68 
                 0.2 
               
               
                   
                   
                   
                   
                   
                   
                 (49) 
                 (32) 
                 (19) 
                   
                   
                   
                   
               
               
                 12A ΔL-ldh2 
                 1.01 
                 8.3 
                 53.1 
                 5.8 
                 111.8 
                 105.0, 
                 3.2, 
                 1.6, 
                 2.0, 
                 BQL 
                 95 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (94) 
                 (3) 
                 (1) 
                 (2) 
                   
                   
                   
               
               
                 12A ΔL-ldh3 
                 1.02 
                 8.0 
                 52.7 
                 9.0 
                 110.2 
                 103.0, 
                 3.2, 
                 2.1, 
                 1.9, 
                 BQL 
                 90 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (94) 
                 (3) 
                 (2) 
                 (2) 
                   
                   
                   
               
               
                 12A ΔD-ldh 
                 1.02 
                 7.7 
                 51.9 
                 BQL 
                 112.4 
                 103.5, 
                 5.4, 
                 1.1, 
                 2.4, 
                 BQL 
                 108 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (92) 
                 (5) 
                 (1) 
                 (2) 
                   
                   
                   
               
               
                 12A ΔD-hic 
                 1.26 
                 7.9 
                 51.5 
                 BQL 
                 112.0 
                 109.7, 
                 BQL 
                 0.5, 
                 1.8, 
                 BQL 
                 109 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (98) 
                   
                 (1) 
                 (2) 
                   
                   
                   
               
               
                 12A ΔL-ldh1/ 
                 1.26 
                 7.1 
                 52.8 
                 15.0 
                 86.7 
                 32.1, 
                 42.6, 
                 12.0, 
                 BQL 
                 BQL 
                 64 
                 0.2 
               
               
                 ΔD-ldh 
                   
                   
                   
                   
                   
                 (37) 
                 (49) 
                 (14) 
                   
                   
                   
                   
               
               
                 12A ΔL-ldh1/ 
                 1.11 
                 9.7 
                 51.2 
                 13.9 
                 79.8 
                 64.9, 
                 BQL 
                 14.9, 
                 BQL 
                 BQL 
                 61 
                 0.2 
               
               
                 ΔD-hic 
                   
                   
                   
                   
                   
                 (81) 
                   
                 (19) 
                   
                   
                   
                   
               
               
                 12A ΔL-ldh1/ΔL- 
                 0.93 
                 9.4 
                 52.5 
                 10.3 
                 71.4 
                 BQL 
                 51.5, 
                 18.6, 
                 BQL 
                 1.3, 
                 57 
                 0.4 
               
               
                 ldh2/ΔD-ldh 
                   
                   
                   
                   
                   
                   
                 (72) 
                 (26) 
                   
                 (2) 
                   
                   
               
               
                 12A ΔL-ldh1/ΔL- 
                 0.52 
                 31.3 
                 21.7 
                 12.8 
                 36.1 
                 0.6, 
                 0.4 
                 7.6, 
                 7.2, 
                 20.3, 
                 52 
                 7.6 
               
               
                 ldh2/ΔD-hic 
                   
                   
                   
                   
                   
                 (2) 
                 (1) 
                 (21) 
                 (20) 
                 (56) 
                   
                   
               
               
                 12A (pTRKH2) 
                 1.01 
                 13.2 
                 52.5 
                 BQL 
                 108.9 
                 100.4, 
                 7.6, 
                 BQL 
                 0.9, 
                 BQL 
                 104 
                 0.0 
               
               
                   
                   
                   
                   
                   
                   
                 (92) 
                 (7) 
                   
                 (1) 
                   
                   
                   
               
               
                 12A (pPGM-PET) 
                 0.95 
                 6.79 
                 51.3 
                 8.2 
                 95.1 
                 14.8, 
                 13.2, 
                 58.1, 
                 9.0, 
                 BQL 
                 80 
                 2.1 
               
               
                   
                   
                   
                   
                   
                   
                 (16) 
                 (14) 
                 (61) 
                 (10) 
                   
                   
                   
               
               
                 12A ΔL-ldh1 
                 1.11 
                 11.3 
                 52.3 
                 2.7 
                 87.7 
                 41.6, 
                 34.0, 
                 12.1, 
                 BQL 
                 BQL 
                 80 
                 0.2 
               
               
                 (pTRKH2) 
                   
                   
                   
                   
                   
                 (47) 
                 (39) 
                 (14) 
                   
                   
                   
                   
               
               
                 12A ΔL-ldh1 
                 1.03 
                 6.8 
                 51.0 
                 16.3 
                 102.1 
                 2.7, 
                 5.0, 
                 84.5, 
                 9.8 
                 BQL 
                 76 
                 10.9 
               
               
                 (pPGM-PET) 
                   
                   
                   
                   
                   
                 (3) 
                 (5) 
                 (83) 
                 (10) 
                   
                   
                   
               
               
                 12A ΔL-ldh1/ΔL- 
                 1.01 
                 7.9 
                 50.9 
                 16.0 
                 100.2 
                 0.7, 
                 5.1, 
                 85.3, 
                 9.1, 
                 BQL 
                 75 
                 14.7 
               
               
                 ldh2 (pPGM-PET) 
                   
                   
                   
                   
                   
                 (1) 
                 (5) 
                 (85) 
                 (9) 
               
               
                   
               
               
                   a Reported by the initial concentration of glucose or citrate subtracted by the final concentration of the respective compound at 48 hrs. 
               
               
                   b In parenthesis, metabolic end product distribution by % of total. 
               
               
                   c Calculated by percentage of total metabolic end products produced/2 × (glucose + citrate) in mmoles. 
               
               
                   d Expressed as molar ratio, where lactate is the summation of both the L- and D- forms. 
               
               
                 Abbreviations: BQL = below quantifiable level; NA = not applicable; Glu = glucose; Cit = citrate; Lac = lactate; ETOH = ethanol; Ace = acetate; Pyr = pyruvuate. 
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
       Example D 
     Conversion of a Lactic Acid Bacterium  Lactobacillus casei  12A to an Ethanologen 
       [0053]      Lactobacillus casei  12A was selected as the biofuels parental strain based upon its alcohol tolerance (grows in the presence of &gt;10% ethanol), carbohydrate utilization, and relatively high transformation efficiency. This organism metabolizes hexoses through the Embden-Meyerhof-Parnas pathway and converts pyruvate to lactate via a variety of different enzymes; including four L-lactate dehydrogenases (Ldh), one D-Ldh, and one D-hydroxyisocaproate dehydrogenase. 
         [0054]    Essential characteristics of organisms to be utilized for microbial production of ethanol from plant biomass include the ability to secrete enzymes, transport glucose and xylose, metabolize glucose and xylose to ethanol, as well as have sufficient ethanol tolerance to make the fermentation economically viable. It is unlikely an organism capable of meeting all of these criteria will be isolated from nature. Therefore, rational strategies to engineer strains for the industrial production of ethanol from plant biomass are preferred. The following characteristics make  L. casei  12A an ideal Gram-positive species for research in this area:
       Designation as a GRAS (Generally Regarded As Safe) species.   Established platforms for introducing and expressing foreign DNA.   Relatively simple fermentative metabolism with almost complete separation of cellular processes for biosynthesis and energy metabolism.   Resistance to environmental stress, including high concentrations of acids and biofuels   Ability to use lignocellulosic carbohydrates.   Ability to secrete and display proteins, hence potential for use in consolidated bioprocessing.       
 
         [0061]    We pursued two strategies concurrently to redirect  L. casei  12A fermentation to ethanol. The first strategy involved inactivation of enzymes that consume pyruvate under anaerobic conditions without producing ethanol, including the D-Ldh; four L-Ldhs; D-(D-Hic); acetolactate synthase (Als); and oxaloacetate decarboxylase (Oad). This approach has been used to inactivate L-ldh1, L-ldh2, and D-hic, as well as to construct the L-ldh1/L-ldh2, double mutant. The highest level of ethanol formation was observed with the ΔL-ldh1/ΔL-ldh2 double mutant, which produces ethanol as 14% of its metabolic end products. 
         [0062]    Our second strategy for increasing flux to ethanol involved expressing ethanol producing enzymes. A codon optimized “PET” cassette comprised of the  Zymomonas mobilis  genes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh2) was constructed, and placed under the control of the  L. casei  12A pgm promoter, pgm ribosomal binding site and kdgR transcriptional terminator. When this construct was introduced into  L. casei  12A, ethanol made up 61% of metabolic end products formed. When introduced into  L. casei  12A (ΔL-ldh1), ethanol was the dominant product observed (91% of metabolic end productions). Results from this analysis indicate that the two approaches are complementary and demonstrate that redirecting metabolic flux in  L. casei  from lactate to an alcohol can be readily achieved. 
         [0063]    The general strategy that was used to redirect metabolic flux in  L. casei  12A from lactic acid to ethanol is illustrated in detail in  FIG. 3 . Two different methods were used to carry out the strategy. The first method, involving gene deletion, is illustrated in  FIG. 4 . The second method, involving the construction and subsequent expression of a synthetic PET expression cassette construct in pTRKH2, is illustrated in  FIG. 5 . The growth of the resulting  L. casei  12A ethanologens in Chemically Defined Medium (CDM) is illustrated in  FIG. 6 . The fermentation by-products of the  L. casei  mutants grown in CDM were measured, and the results are shown in Table 6. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Fermentation products of  L. casei  12A and mutants with and 
               
               
                 without pTRKH2 or pPGM-PET growth in CDM for 48 hrs. 
               
             
          
           
               
                 Derivative 
                 Ethanol (%) 
                 L-Lactate (%) 
                 D-Lactate (%) 
               
               
                   
               
             
          
           
               
                 12A 
                 0.0 
                 95.0 
                 5.0 
               
               
                 12A ΔL-ldh1 
                 6.0 
                 49.0 
                 45.0 
               
               
                 12A ΔL-ldh2 
                 0.0 
                 96.0 
                 4.0 
               
               
                 12A ΔD-hic 
                 0.0 
                 71.0 
                 29.0 
               
               
                 12A ΔL-ldh1ΔL-ldh2 
                 14.0 
                 34.0 
                 52.0 
               
               
                 12A (pTRKH2) 
                 0.0 
                 95.0 
                 5.0 
               
               
                 12A (Ppgm-PET) 
                 61.0 
                 34.0 
                 1.0 
               
               
                 12A ΔL-ldh1 (pTRKH2) 
                 13.0 
                 47.0 
                 40.0 
               
               
                 12A ΔL-ldh1 
                 90.9 
                 1.5 
                 1.5 
               
               
                 (Ppgm-PET) 
               
               
                   
               
               
                 Note: 
               
               
                   L. casei  12A mutants were grown in MRS from glycerol stock for 24 hrs at 37° C. then transferred to MRS and incubated for an additional 18 hrs. CDM containing 50 mM glucose was inoculated and incubated in GC vials for 48 hrs at 37° C. At the 48-hr time point, supernanant was drawn off and submitted to GLBRC enabling technologies for fermentation by-product analysis via HPLC-RID. 
               
             
          
         
       
     
         [0064]    Conclusions. 
         [0065]    Inactivation of L-Ldh1 reduced flux towards L-lactate and enhanced flux towards D-lactate and ethanol. Inactivation of L-Ldh2 increased these changes in metabolic flux. 
         [0066]    In  L. casei  12A with the PET cassette, ethanol made up 61% of metabolic end products formed, while 91% of metabolic end productions were directed to ethanol when the PET cassette was introduced into  L. casei  12A ΔL-ldh1. 
         [0067]    The two pronged strategy, inactivating genes encoding enzymes that produce lactic acid and introducing the PET cassette, effectively converted  L. casei  12A from producing lactate as its main metabolic product to producing ethanol as its main metabolic end product. 
       REFERENCES 
       [0000]    
       
         Cai, H., Thompson, R. L., Broadbent, J. R., and Steele, J. L. (2009). Genome Sequence and Comparative Genome Analysis of  Lactobacillus casei : Insights into their Niche-associated Evolution. Genome Biol. and Evol. 1:239-257. 
         Duong, T., Miller, M. J., Barrangou, R., Azcarate-Peril, M. A., and Klaenhammer, T. R. (2010). Construction of vectors for inducible and constitutive gene expression in  Lactobacillus . Microbiol Biotech, 4(3): 357-367. 
         Kristich, C. J., Chandler, J. R., and Dunny, G. M. (2007). Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of  Enterococcus faecalis . Plasmid 57:131-144. 
       
     
       Example E 
     Use of Alternate Promoter 
       [0071]    In the previous examples, a first generation  Lactobacillus casei  ethanologen was created by a two pronged approach to redirect metabolic flux in  L. casei  12A from lactate to ethanol. The first prong was to inactivate genes encoding lactate dehydrogenases, enzymes which compete with the 12A pathway to ethanol. The second prong was the introduction of the genes from  Zymomonas mobilis  that encode pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (Adh2) activities (PET cassette). These genes were designed utilizing the  L. casei  codon usage for highly expressed genes and placed under the control of  L. casei  phosphoglycerate mutase promoter, thought to be a constitutively expressed promoter. 
         [0072]    This approach was highly successful, resulting in a strain that utilized 504.5 mM glucose (9.1%) glucose in 96 h and produced 934.7 mM of “pyruvate-derived” metabolic end products, which is 92.6% of the theoretical yield from 504.5 mM glucose in a 500 ml fermentation vessel under anaerobic conditions at 37° C. in a defined media with 540 mM glucose. Ethanol was produced at a level of 771.3 mM (4.5%), which was 82.5% of the metabolic end-products. The second most abundant metabolic end product was pyruvate which was present at 110.1 mM after 96 h. 
         [0073]    Pyruvate accumulation began at approximately 21 h. At the same time, ethanol as a percentage of the total metabolic end products began to decrease (% ethanol in total), suggesting that pyruvate decarboxylase activity becomes limiting at that time. This corresponds to the entry of this organism into stationary phase, suggesting that the  L. casei  phosphoglycerate mutase (pgm) promoter used to drive expression of the PET cassette is poorly expressed in stationary phase. It is highly likely that pyruvate accumulation can be overcome by utilizing a  L. casei  promoter highly expressed in stationary phase. 
         [0074]    Accordingly, this prophetic example discloses the next generation  L. casei  ethanologen, having the PET cassette placed under the control of a promoter that is highly expressed in stationary phase. For example, either the GroEL or DnaK promoters, as they have been demonstrated to be highly expressed in a related organism,  L. plantarum , when this organism was exposed to ethanol (Gyu et al. 2012). The anticipated result from such a construct would be that the 110.1 mM pyruvate that was observed to accumulate in the previous fermentation (see above) would be converted to ethanol. This would then yield 881.4 (110.1+771.3) mM of ethanol, or 87.4% of the theoretical yield from 504.5 mM glucose. 
       REFERENCES 
       [0000]    
       
         Lee, S. G., K. W. Lee, T. H. Park, J. Y. Park, N. S. Han, and J. H. Kim. 2012. Proteomic analysis of proteins increased or reduced by ethanol of  Lactobacillus plantarum  ST4 isolated from  makgeolli , traditional Korean rice wine. J. Microbiol. Biotechnol. 22:516-525.

Technology Classification (CPC): 2