Galactose utilization

Abstract

The present disclosure describes a genetically engineered bacteria that relieves the catabolite repression problem exerted by the Spot 42 small regulatory RNA by adding a galactokinase that does not contain the Spot 42 binding region. As such, galK and galM and the like can be expressed allow better galactose utilization.

Claims

1. A recombinant bacteria, said bacteria being from a gammaproteobacterial species having a Spot 42 (spf) responsive galactokinase (GalK) when said species is wild type at GalK, and said bacteria comprising: a) a knockout mutation of an endogenous spot 42 responsive GalK; and b) expressing an open reading frame (ORF) from a GalK lacking a Spot 42 binding region (GalK.sup.Spot42-) under the control of a promotor that is not native to the GalK.sup.Spot42-; wherein said recombinant bacteria is able to avoid catabolite repression and grow on mixed sugars comprising galactose and glucose.

2. The recombinant bacteria of claim 1, wherein said mixed sugars comprising galactose and glucose comprises soymeal hydrolysate.

3. The recombinant bacteria of claim 1, said ORF being operably linked to an expression vector.

4. The recombinant bacteria of claim 1, said ORF being operably linked to an inducible expression vector.

5. The recombinant bacteria of claim 1, said ORF being operably linked to an constitutive expression vector.

6. The recombinant bacteria of claim 1, said species being selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Escherichia, Shigella, Klebsiella, Salmonella, Yersinia, Vibrio, Aliivibrio, Photobacterium and Grimontia.

7. The recombinant bacteria of claim 1, said GalK.sup.Spot42- being an ORF selected from GalK.sup.Spot42- genes from Bifidobacterium longum subsp. infantis, Arabidopsis thaliana, Trichoderma reesei, Streptococcus pneumonia, Streptococcus thermophilus and Saccharomyces cerevisiae.

8. The recombinant bacteria of claim 1, said bacteria further comprising an overexpressed gal operon.

9. The recombinant bacteria of claim 1, said bacteria further comprising an expression vector that encodes a gal operon.

10. The recombinant bacteria of claim 1, said bacteria further comprising an inducible expression vector that encodes a gal operon.

11. The recombinant bacteria of claim 1, said bacteria further comprising overexpression of GalE, GalT, GalK, GalP and pgm.

12. The recombinant bacteria of claim 1, said bacteria further comprising an inducible expression vector encoding E. coli GalE, GalT, GalK, GalP and pgm.

13. A method of producing a fatty acid, comprising growing the recombinant bacteria of claim 1 in a culture medium comprising galactose and glucose for a time sufficient to produce a fatty acid, and isolating said fatty acid from bacteria, or said culture medium, or both.

14. A method of producing a fatty acid, comprising growing the recombinant bacteria of claim 11 in a culture medium comprising galactose and glucose for a time sufficient to produce a fatty acid, and isolating said fatty acid from bacteria, or said culture medium, or both.

15. A recombinant bacteria, said bacteria being a gammaproteobacterial species having a Spot 42 (spf) responsive GalK when said species is wild type at GalK, and said recombinant bacteria comprising: a) a knockout mutation of an endogenous spot 42 responsive GalK; and b) expressing a GalK.sup.Spot42- ORF from Bifidobacterium under a promoter that is not native to Bifidobacterium; wherein said bacteria is able to avoid catabolite repression and grow on mixed sugars comprising galactose and glucose.

16. The recombinant bacteria of claim 15, said bacteria further comprising overexpression of GalE, GalT, GalK, GalP and pgm.

17. A method of producing a fatty acid, comprising growing the recombinant bacteria of claim 16 in a culture medium comprising galactose and glucose for a time sufficient to produce a fatty acid, and isolating said fatty acid from bacteria, or said culture medium, or both.

18. The method of claim 17, wherein said culture medium comprises soymeal hydrolysate.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: Diagram showing the gal operon. The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. The operon contains two operators, O.sub.E (for external) and O.sub.1. The former is just before the promoter, and the latter is just before the galE gene (the first gene in the operon).

(2) FIG. 2: Three gal operon and the mRNA species detected by Northern blot analysis (2) are presented as thick and thin lines, respectively. The numbers indicate the positions of the stop codon of each cistron from the transcription initiation sites of the P1 promoter. The left-right arrows (.Math.) indicate the primer sets for RT-qPCR. Note that the primer sets are at the 3′ end of the respective genes, except in the case of galT. The stem and loop structure for the transcription termination of mM1 is presented at the end of the operon. The three short lines under the mK1, mM1, and mK2 mRNA species indicate the regions where Spot 42 binds. The sequence of Spot 42 and its binding site at the galT-galK cistron junction are shown with short vertical lines indicating base-pairing. The stop codon of galT and the start codon of galK are indicated in bold case. Hfq binds to the 3′ end of Spot 42. The numbers indicate nucleotide positions. From Wang (2015).

(3) FIG. 3: Spot 42 RNA. Spot 42 can interact directly with mRNA targets through base pairing. Although now known to bind at least 14 different operons, the first Spot 42 targets a short complementary region at the translation initiation region of galK (encodes a galactokinase). galK is the third gene in the galactose operon, which contains four genes (galETKM) and produces a polycistronic mRNA. Spot 42 mediates discoordinate expression of the gal operon (i.e., the individual genes in the operon are not similarly expressed) by binding to the galK Shine-Dalgarno region, thereby blocking ribosome binding and translation of the galK gene. Data suggests that Spot 42 plays a role in fine-tuning gene expression to optimize the utilization of carbon sources.

(4) FIG. 4-8: Diagrams showing the various vector constructs used herein.

(5) FIG. 4: Plasmid pPL18-gal/bi_galK uses pTrc99a as backbone, carrying an acyl-ACP thioesterase gene from Ricinus communis, and five genes involved in galactose utilization (galP, pgm, galE, galT, galM) from E. coli and bi_galK from Bifidobacterium infantis.

(6) FIG. 5: pPL18-gal/bi_galK uses pTrc99a as backbone, carrying an acyl-ACP thioesterase from Ricinus communis and fabZ, galP, pgm, galE, galT, galM, galK from E. coli.

(7) FIG. 6: pTrc-bigalK was constructed from pTrc99a, carrying bigalK from Bifidobacterium infantis

(8) FIG. 7: pTrc-gal operon was constructed from pTrc99a, carrying galE, galT, galM, galK from E. coli

(9) FIG. 8: pTrc-gal/bi_galK was constructed from pTrc99a, carrying galE, galT, galM from E. coli and bi_galK from Bifidobacterium infantis.

DETAILED DESCRIPTION

(10) The invention provides a novel method of making any bacterial product, utilizing a recombinant bacteria that has an added, exogenus Spot 42 negative galactokinase gene therein or wherein the endogenous gene has been modified to be spot 42 negative. The galK and galM in the gal operan can then be expressed normally and the GalK enzyme can be expressed even under repressed conditions.

(11) The present described recombinant bacteria are exemplified with respect to the E. Coli strains listed in Table 1 and Bacillus subtilis. However, this is exemplary only, and the invention can be broadly applied to any bacteria strain that is applied in any species having a spf gene in its native or wild type state. The spf gene is highly conserved in Escherichia, Shigella, Klebsiella, Salmonella, Yersinia genera within the Enterobacteriaceae family. In E. coli the spf gene is flanked by polA (upstream) and yihA (downstream). A CRP binding sequence and −10 and −35 promoter sequences are found upstream of spf.

(12) Spf is also highly conserved within the Vibrionaceae family, and was recently identified in all 76 available Vibrionaceae genomes (e.g., Vibrio, Aliivibrio, Photobacterium and Grimontia genera). In e.g., Vibrio cholerae, Vibrio vulnificus, Aliivibrio fischeri and Aliivibrio salmonicida the spf gene is flanked by polA (upstream) and a sRNA gene encoding the novel VSsRNA24 (downstream).

(13) TABLE-US-00002 TABLE 1 Strains and gene information used in the examples Strain Description or genotype E. coli MG1655 Wild type E. coli ML103 MG1655 ΔfadD E. coli ML190 MG1655 ΔfadDΔptsG E. coli XZK009 MG1655 ΔfadDΔptsGΔspf E. coli SL103 MG1655 ΔfadDΔgalR E. coli SL190 MG1655 ΔfadDΔptsGΔgalR

(14) Exemplary vector constructs are shown in FIG. 4-8.

(15) The following examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.

(16) Briefly, we constructed a plasmid named pTrc-gal/bi_galK that contained a galactokinase from Bifidobacterium longum subsp. infantis (bi_galK), which does not contain the Spot 42 binding region. This plasmid is an example of the type of exogenuous genes that can replace the native E. coli galK. This plasmid construct also carries the E. coli galE, galT and galM genes.

(17) The use of a galactokinase from Bifidobacterium longum subsp. infantis (bi_galK) is just an example of the type of plasmid that can be created to combine with the bacteria strain. Other similar galK genes without the Spot 42 region can be used, such as those list in the Table 2. Further, as noted an endogenous gene can be gene edited to remove or mutate the Spot 42 binding region.

(18) TABLE-US-00003 TABLE 2 Some examples of Spot 42-negative galactokinases (galK) from the following organisms that can be used, in addition to Bifidobacterium infantis, to replace E. coli galK GeneBank Access Gene Organism NO or Gene ID atgalk Arabidopsis thaliana 819837 gal1 Trichoderma reesei AY249022 galKSpe4 Streptococcus pneumoniae AAK75925 galK Streptococcus thermophilus AAU21544 gal1 Saccharomyces cerevisiae 852308

(19) To demonstrate the effectiveness of using a galactokinase that does not contain the Spot 42 binding region for galactose utilization in engineered bacteria, we performed experiments using this pTrc-gal/bi_galK construct. We found that strains carrying the pTrc-gal/bi_galK outperform the galactose utilization in those bacteria carrying the native galK both in galactose only medium, as well as in medium containing a mixture of sugars (glucose and galactose).

(20) Additionally, using fatty acid as the targeted product, strains carrying the pTrc-gal/bi_galK produce more fatty acids than those carrying the native galK in galactose only medium and in soymeal hydrolysate, which contains galactose as a major sugar component.

(21) The production of fatty acids as an exemplary target product is an example of one means to monitor the abilities of the recombinant bacteria, but the same technology can be used in producing other products from galactose. A more detailed description of our experiments are below.

Cultivation Method

(22) First, we cultivated a colony of cells with the Spot 42 negative galK gene. A single colony of strain MG1655 (pTrc99a), MG1655 (pTrc-bi_galK), MG1655 (pTrc-gal operon) or MG1655 (pTrc-gal/bi_galK) was inoculated into 5 ml of Luria-Bertani (LB) and incubated in an orbital shaker operated at 250 rpm at 37° C. overnight. The preculture was inoculated into a flask containing 50 mL of the culture medium with 1% (v/v) inoculum. The culture medium contained: tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, galactose 15 g/L, ampicillin 100 μg/L, pH 7.5 supplemented with 1 mM IPTG.

(23) Shake flask experiments were performed at 30° C. with shaking at 250 rpm for 72 h. The samples were taken at 24 and 48 hours after inoculation. Galactose utilization (g/L) was monitored using an HPLC. Fatty Acid production levels were also monitored using GC or GC/MS.

(24) These cultivation conditions were generally employed throughout the experiments, with modification as noted.

Bi_Galk & Galactose Utilization

(25) Four strains, MG1655 (pTrc99a), MG1655 (pTrc-bi_galK), MG1655 (pTrc-gal operon) or MG1655 (pTrc-gal/bi_galK) were examined for their ability to utilize galactose. In addition, the effect of different inducer (IPTG) concentration on galactose utilization for this series of plasmids was studied to determine the optimal induction level. The results for these experiments are summarized in Table 3.

(26) All strains showed the highest galactose utilization at the end of 48 hours at 0.025 mM IPTG. The strain carrying the plasmid pTrc-gal/bi_galK (a Spot 42 negative galactokinase) consumed 10.51 g/L of galactose. This is about 10% better than the strain carrying the plasmid pTrc-gal operon expressing the native E. coli galactose operon.

(27) Our experimental data indicated that the optimal induction level is around 0.025 mM IPTG. Further, replacing the native E. Coli GalK with a GalK from a Bifidobacterium infantis (bi_galK) that lacks the Spot 42 binding region improved galactose utilization by more than 10%.

(28) TABLE-US-00004 TABLE 3 Galactose utilization under different IPTG concentrations in MG1655 harboring different plasmid constructs Galactose utilization (g/L) Strain Relevant genotype IPTG (mM) 24 h 48 h bi_galK+: overexpression of galK from Bifidobacterium infantis in pTrc99a ec_GalETKM+: overexpression of galETKM from Escherichia coli in pTrc99a MG1655 (pTrc- bi_GalK+ 0 3.07 2.81 bi_galK) 0.025 1.79 5.33 0.05 1.87 1.87 0.1 — — 0.2 1.67 1.16 MG1655 ec_GalE+ 0 4.59 7.12 (pTrc-gal operon) ec_GalT+ 0.025 3.60 7.91 ec_GalK+ 0.05 3.20 7.09 ec_GalM+ 0.1 2.77 6.36 0.2 2.11 5.16 MG1655 ec GalE+ 0 4.38 6.65 (pTrc-gal/bi_galK) ec GalT+ 0.025 4.09 10.51 ec GalM+ 0.05 3.89 9.82 bi_GalK+ 0.1 3.67 9.94 0.2 3.35 8.53

Bi_Galk & Different Genetic Backgrounds

(29) A series of experiments were also performed with two different host E. coli strains to demonstrate that the genetic background was not controlling. Strain ML190 is a ptsG mutant and strain XZKO09 is a ptsG, spf double mutant. The results are summarized in Table 4.

(30) TABLE-US-00005 TABLE 4 Galactose utilization by a series of gal constructs in host strain ML190, SL190 and XZK009 Galactose utilization (g/L) Strain Relevant genotype IPTG (mM) 24 h 48 h Bi_galK+: overexpression of galK from Bifidobacterium infantisin pTrc99a ec_GalETKM+: overexpression of galETKM from Escherichia coli in pTrc99a ML190 (pTrc99a) ΔfadDΔptsG 0.025 3.05 4.01 ML190 (pTrc- bi_galK+ 1.67 6.65 bi_galK) ML190 (pTrc-gal ΔfadDΔptsG 2.62 7.17 operon) ec GalE+ ec GalT+ eccGalK+ ec GalM+ ML190 (pTrc- ΔfadDΔptsG 1.59 9.92 gal/bi_galK) ec GalE+ ec GalT+ ec GalM+ bi_galK+ SL190 (pTrc99a) ΔfadDΔptsGΔgalR 0.025 0 1.80 SL190 (pTrc- ΔfadDΔptsGΔgalR 0 2.09 bi_galK) bi_galK+ SL190 (pTrc-gal ΔfadDΔptsGΔgalR 2.93 1.56 operon) ec GalE+ ec GalT+ ec GalK+ ec GalM+ SL190 (pTrc- ΔfadDΔptsGΔgalR 1.54 0.98 gal/bi_galK) ec GalE+ ec GalT+ ec GalM+ bi_galK+ XZK009 (pTrc99a) ΔfadDΔptsGΔspf 0.025 2.78 3.98 XZK009 (pTrc- ΔfadDΔptsGΔspf 1.28 6.34 bi_galK) bi_galK+ XZK009 (pTrc-gal ΔfadDΔptsGΔspf 0.81 6.97 operon) ec GalE+ ec GalT+ ec GalK+ ec GalM+ XZK009 (pTrc- ΔfadDΔptsGΔspf 7.11 11.71 gal/bi_galK) ec GalE+ ec GalT+ ec GalM+ bi_galK+

(31) Several observations can be made from the results in Table 4: The ML190 (pTrc99a), carrying the cloning vector pTrc99a (see GenBank| M22744), utilized 4.01 g/L of galactose at 48 h; the ptsG, spf double mutant strain, XZK009 (pTrc99a), does not increase galactose utilization (3.98 g/L). For the ptsG mutant strain ML190, overexpression of bi_galK only or the native gal operon, or replacing the native E. Coli GalK with bi_galK all improved galactose utilization from utilized 4.01 g/L to a high value of 9.92 g/L by the ML190 (pTrc-gal/bi_galK) strain, more than doubling the galactose utilization.

(32) Similar observations were observed for the spf mutant strain XZKO09. Overexpression of bi_galK only or the native gal operon, or replacing the native ec GalK with bi_galK all improved galactose utilization from utilized 3.98 g/L to a high value of 11.71 g/L by the XZKO09 (pTrc-gal/bi_galK) strain, representing a tripling of the galactose utilization.

(33) Thus, the experimental data indicated that replacing the native E. Coli GalK with a galactokinase without the Spot 42 binding region (such as galK from Bifidobacterium infantis—bi_galK) improves galactose utilization significantly. Additional deactivation of the Spot 42 gene (spf) further enhances the galactose utilization.

Bi_Galk & Mixed Sugars

(34) A series of experiments were performed with the host strain ML190 carrying plasmids pTrc-bi_galK, pTrc-gal operon, and pTrc-gal/bi_galK. These experiments are designed to examine the ability of these strains to utilize galactose in a sugar mixture and the results are shown in Table 5. The ability to utilize galactose from a sugar mixture is highly desirable because many cheap or recycled feedstocks are a mix of sugars.

(35) TABLE-US-00006 TABLE 5 Galactose utilization of mixed sugar medium by ML190 carrying gal series plasmids Relevant IPTG Galactose utilization (g/L) Strain genotype (mM) 24 h 48 h 72 h Bi_galK+: overexpression of galK from Bifidobacterium infantis in pTrc99a ec GalETKM+: overexpression of GalETKM from Escherichia coli in pTrc99a ML190 (pTrc- ΔfadDΔptsG 0.025   0/3.04 0.78/6.51 1.68/6.38 bi_galK) bi_galK+ ML190 (pTrc- ΔfadDΔptsG 0.27/1.9  1.75/4.96 3.40/6.78 gal operon) ec GalE+ ec GalT+ ec GalK+ ec GalM+ ML190(pTrc- ΔfadDΔptsG 0.72/3.19 3.31/6.97 6.57/6.99 gal/bi_galK) ec GalE+ ec GalT+ ec GalM+ bi_galK+

(36) In this set of experiments, the ML190 (pTrc-gal/bi_galK) strain performed the best. The experimental data in Table 5 indicated that replacing the native E. Coli galK with a galK from Bifidobacterium infantis (bi_galK) improves galactose utilization significantly.

Bi_Galk & Improved Productivity from Galactose

(37) Another means of characterizing the recombinant bacteria disclosed herein is by monitoring their ability to form or synthesize certain products. Here, we used medium chain length fatty acids as a marker for characterizing the improvement in productivity over native or wild type bacteria.

(38) The synthesis of medium chain length fatty acids was used to demonstrate the use of bi_galK to improve galactose utilization and fatty acid production. SL103 was used as the host strain.

(39) Three plasmids were examined, plasmid pXZ18 carrying only an acyl-ACP thioesterase from Ricinus communis (rc TE), plasmid pPL18-gal carrying the galactose operon from E. coli in addition to the rc TE, and plasmid pPL18-gal/bi_galK carrying the galactose operon from E. coli with the GalK replaced by bi_galK in addition to the rc TE. The results are summarized in Table 6.

(40) TABLE-US-00007 TABLE 6 Fatty acid production by strain SL103 in galactose Concentration of total Relevant IPTG fatty acid (g/L) Galactose utilization (g/L) Strain genotype (mM) 24 h 48 h 72 h 24 h 48 h 72 h SL103 rc_TE.sup.+ 0.025 0.79 1.03 1.06 5.91  8.27 10.02 (pXZ18) SL103 ΔfadDΔgalR 0.95 1.24 1.23 2.55/ 7.43 7.98 (pPL18-gal) ec GalE+ ec GalT+ ec GalK+ ec GalM+ ec GalP+ ec Pgm+ SL103 ΔfadDΔgalR 0.33 1.82 2.10 0.58/ 7.87 12.45 (pPL18- ec GalE+ gal/bi_galK) ec GalT+ bi_galK+ ec GalM+ ec GalP+ ec Pgm+ rc_TE.sup.+: overexpression of acyl-ACP thioesterase from Ricinus communis under the PTRC promoter in pTrc99a bi_galK+: overexpression of bi_galK from Bifidobacterium infantis in pTrc99a ec GalETKM+: overexpression of galETKM from Escherichia coli in pTrc99a ec GalP+: overexpression of galP from Escherichia coli in pTrc99a ec Pgm+: overexpression of pgm from Escherichia coli in pTrc99a

(41) The strain SL103 (pXZ18) served as the control and it produced 1.02 g/L of fatty acids at 72 h. The SL103 (pPL18-gal) strain with overexpression of the native galactose operon improved the fatty acid production by about 20% to 1.23 g/L. The SL103 (pPL18-gal/bi_galK) strain with the bi_galK improved the fatty acid production significantly; a two-fold increase to 2.10 g/L was obtained when compared with the control strain, SL103 (pXZ18).

(42) Thus, this set of experiments, using production of fatty acids as an exemplary product, demonstrated that the use of a galactokinase without the Spot 42 binding region can significantly improve product production. Again, other fatty acids or synthesis products can also be used as benchmarks to monitor the improvement of galactose utilization.

Bi_Galk & Soymeal Hydrolysate

(43) To determine how well the recombinant bacteria were able to utilize galactose from a sugar mixture, a soymeal hydrosolyate was added to the culture process.

(44) Carbohydrates from soymeal hydrolysate provide an inexpensive carbon source. Further, soymeal hydrolysate contains a mixture of sugars, but the major components are glucose, fructose and galactose.

(45) As before, the synthesis of medium chain length fatty acids was used as a benchmark to demonstrate the use of bi_galK to improve galactose utilization and fatty acid production from soymeal hydrolysate. Similar to above, SL103 was used as the host strain. Three plasmids were examined, plasmid pXZ18 carrying only an acyl-ACP thioesterase from Ricinus communis (rc TE), plasmid pPL18-gal carrying the galactose operon from E. coli in addition to the rc TE, and plasmid pPL18-gal/bi_galK carrying the galactose operon from E. coli with the galK replaced by bi_galK in addition to the rc TE. The results are summarized in Table 7.

(46) TABLE-US-00008 TABLE 7 Fatty acid production by strain SL103 using soymeal hydrolysate Concentration of Relevant total fatty acid (g/L) Strain genotype IPTG (mM) 24 h 48 h 72 h rc_TE.sup.+: overexpression of acyl-ACP thioesterase from Ricinus communis under the Ptrc promoter in pTrc99a bi_galK+: overexpression of bi_galK from Bifidobacterium infantis in pTrc99a ec GalE+: overexpression of galE from Escherichia coli in pTrc99a ec GalT+: overexpression of galT from Escherichia coli in pTrc99a ec GalK +: overexpression of galK from Escherichia coli in pTrc99a ec GalM +: overexpression of galM from Escherichia coli in pTrc99a ec GalP +: overexpression of galP from Escherichia coli in pTrc99a ec Pgm +: overexpression of pgm from Escherichia coli in pTrc99a SL103 (pXZ18) ΔfadDΔgalR 0.025 0.64 1.43 1.36 rc_TE.sup.+ SL103 (pPL18-gal) ΔfadDΔgalR 0.49 0.65 1.07 ec GalE+ ec GalT+ ec GalK+ ec GalM+ ec GalP+ ec Pgm+ SL103 (pPL18- ΔfadDΔgalR 1.02 1.57 1.61 gal/bi_galK) ec GalE+ ec GalT+ bi_galK+ ec GalM+ ec GalP+ ec Pgm+

(47) The strain SL103 (pXZ18), which served as the control, produced 1.36 g/L of fatty acids at 72 h. The SL103 (pPL18-gal) strain with overexpression of the native galactose operon did not perform well; this strain only produce 1.07 g/L. However, the SL103 (pPL18-gal/bi_galK) strain with the bi_galK improved the fatty acid production to 1.61 g/L, an 18% improvement over that of the control strain, SL103 (pXZ18).

(48) Thus, this set of experiments, using production of fatty acids as an example, further demonstrated that the use of a galactokinase without the Spot 42 binding region can significantly improve product production from soymeal carbohydrate hydrolysate containing a mixture of sugars.

Prophetic: Bacillus

(49) The above experiments were repeated in Bacillus subtilis cells.

(50) The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli-B. subtilis shuttle vector, pMTLBS72, exhibited full structural stability and was used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHT01 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids pAMβ1 and pBS72 can be used. Several other suitable expression systems are available.

(51) Since the GAL genes are ubiquitous, the modified Bacillus performed as expected.

(52) The following references are incorporated by reference in their entirety for all purposes: U.S. Pat. No. 8,906,667 Increasing NADPH-dependent products US20140273114 Bacteria and method for synthesizing fatty acids U.S. Pat. No. 8,795,991 Increasing bacterial succinate productivity US20140212935 Short chain fatty acids from bacteria US20140193867 Microbial odd chain fatty acids U.S. Pat. No. 8,709,753 Native NAD-dependent GAPDH replaced with NADP-dependent GAPDH plus NADK US20140093921 Bacteria and method for synthesizing fatty acids U.S. Pat. No. 8,486,686 Large scale microbial culture method U.S. Pat. No. 8,236,525 Reduced phosphotransferase system activity in bacteria U.S. Pat. No. 7,901,924 Increased bacterial CoA and acetyl-CoA pools U.S. Pat. No. 7,709,261 Recycling system for manipulation of intracellular NADH availability Møller, T., et al., Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon, Genes Dev. 2002 Jul. 1; 16(13): 1696-1706. Lim, H. G., et al., Modular design of metabolic network for robust production of n-butanol from galactose-glucose mixtures, Biotechnology for Biofuels 20158:137 (2015). Vorgias C. E., et al., Overexpression and purification of the galactose operon enzymes from Escherichia coli. Protein Expr Purif. 1991 October-December; 2(5-6):330-8. Wang, X., et al., Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene, Proc. nat. Acad. Sci. 112(24): 7581-7586 (2015).

(53) All GenBank, UniProt accession numbers or gene ID numbers referenced herein are incorporated by reference herein in its entirety for all purposes.