DESCRIPTION

MATERIALS AND METHODS FOR IMPROVED MICROBIAL PRODUCTION

OF ORGANIC COMPOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS

) This application claims the benefit of U.S. Provisional Application Serial No.

60/734,443, filed November 8, 2005. This application is also a continuation-in-part of U.S. Application Serial No. 11/501,137, filed August 8, 2006, which claims the benefit of U.S. Provisional Application Serial Nos. 60/706,887, filed August 10, 2005; 60/761,576, filed January 24, 2006; and 60/799,619, filed May 11, 2006.

GOVERNMENT SUPPORT

The subject invention was made with government support under research projects supported by US DOE-DE FG02-96ER20222 and USDA 00-52104-9704. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Recent trends toward the production of "green" chemicals will require development of innovative synthesis techniques that are highly efficient and cost effective.

Throughout the past decade or more, a number of traditional chemical companies in the United States and Europe have begun to develop infrastructures for the production of compounds using biocatalytic processes. Considerable progress has been reported toward new processes for commodity chemicals such as ethanol (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 "Enteric bacterial catalyst for fuel ethanol production" Biotechnol. Prog. 15:855-866; Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano, K.T. Shanmugam, and L. O. Ingram, 2002 "Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli." Appl. Environ. Microbiol. 68:6263-6272), lactic acid (Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam and L. O. Ingram, 2003 "Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110" Appl. Environ. Microbiol. 69:399-407; Chang, D., S. Shin, J. Rhee, and J. Pan, 1999 "Homofermentative

production of D- or L-lactate in metabolically engineered Escherichia coli RRl" Appl. Environ. Microbiol. 65:1384-1389; Dien, B. S., N. N. Nichols, and R. J. Bothast, 2001 "Recombinant Escherichia coli engineered for the production of L-lactic acid from hexose and pentose sugars" J. Ind. Microbiol. Biotechnol. 27:259-264), 1,3-propanediol (Nakamura, US Patent 6,013,494; Tong, L, H. H. Liao, and D. C. Cameron, 1991 "1,3-propanediol production by Escherichia coli expressing genes from the Klebsiella- pneumoniae-DHA regulon" App. Env. Microbiol. 57:3541-3546), and adipic acid (Niu, W., K. M. Draths, and J. W. Frost, 2002 "Benzene-free synthesis of adipic acid" Biotechnol. Prog. 18:201-211).

In addition, advances have been made in the genetic engineering of microbes for higher value specialty compounds such as acetate, polyketides (Beck, B. J., C. C. Aldrich, R. A. Fecik, K. A. Reynolds, and D. H. Sherman, 2003 "Iterative chain elongation by a pikromycin monomodular polyketide synthase" J. Am. Chem. Soc. 125:4682-4683; Dayem, L. C, J. R. Carney, D. V. Santi, B. A. Pfeifer, C. Khosla, and J. T. Kealey, 2002 "Metabolic engineering of a methylmalonyl-CoA mutase - epimerase pathway for complex polyketide biosynthesis in Escherichia coli." Biochem. 41:5193-5201) and carotenoids (Wang, Chia-wei, Min-Kyu Oh, J. C. Liao, 2000 "Directed evolution of metabolically engineered Escherichia coli for carotenoid production" Biotechnol. Prog. 16:922-926).

Pyruvate and acetate are central intermediates for biosynthesis and energy metabolism in plants, animals and microorganisms. Both compounds are commercially produced and used in a variety of products. Pyruvate serves as a starting material for production of amino acids (L-tryptophan, L-tyrosine and others), pharmaceuticals such as L-DOPA, and agrichemicals (Li et al. 2001). Acetate is an important component of many solvents and plastics, in food products, and as a road de-icer (Berraud 2000, Cheryan et al. 1997, Freer 2002).

Although both compounds can be made from sugars as products of microbial metabolism, petrochemical routes have largely displace microbial production of acetate except for food uses (Kirscher 2003). Microbial production of acetate and pyruvate from sugars is more challenging than anaerobic fermentations that produce ethanol or lactate due to the requirement for an external electron acceptor such as oxygen.

Derivatives of Escherichia coli K- 12 (strain W3110) have been developed for acetate (strain TC36; Causey et al. 2003) and pyruvate (strain TC44; Causey et al. 2004) production from glucose in mineral salts medium.

The use of polylactic acid as a biodegradable alternative for petroleum-based plasties is expanding in many areas including textiles, medical implants, drug carriers, food packaging, and cosmetics (Agrawal 2003; Lee et al. 2004; Ray and Bousmina 2005; Wang 2005). To compete effectively as a commodity feedstock for plastics, further improvements are desirable in both the biocatalysts and lactate purification (Narayanan et al. 2004; Ohara et al. 2001; Vaidya et al. 2005; Wasewar 2005).

Strain SZ 132 is an Escherichia coli B strain capable of rapid D-lactate production in complex medium with 10% (w/v) glucose (Zhou et al, 2005). However, this strain was unable to complete the fermentation of 10% (w/v) glucose in mineral salts medium even after prolonged incubation. Maximum fermentation rates in mineral salts medium with 10% (w/v) glucose were roughly half of those observed with 5% (w/v) glucose, while differences between 5% (w/v) and 10% (w/v) sucrose were less pronounced. Osmotic stress associated with 10% (w/v) sucrose is half that of 10% (w/v) glucose.

E. coli is known to adapt to osmotic stress by increasing intracellular levels of potassium glutamate and trehalose, and accumulating other protective osmolytes such as betaine from the environment when available (Csonka 1989; Kempf & Bremer 1998; Purvis et al. 2005). Osmotic stress activates native pathways for the production of glutamate. The increase in acetyl~P pools in acetate kinase-deficient cells appears to phosphorylate NR ₁ , a transcriptional activator that increases production of glutamine synthetase (Feng et al. 1992). Underwood et al. (2004) reported that volumetric rates of ethanol production were increased in mineral salts medium containing 9% (w/v) xylose by supplementing with large amounts of central metabolites or small amounts of protective osmolytes.

There is a need in the art for improved microbial production techniques, including techniques that enhance the production of useful organic compounds by reducing the adverse osmotic effects from sugars and products.

BRIEF SUMMARY OF THE INVENTION

Osmotic stress due to high concentrations of sugars and fermentation products limits the performance of E. coli in simple batch fermentation, creating a leaky nutritional requirement. This stress-related nutrient requirement can be satisfied, according to the subject invention, by adding an osmoprotectant. Advantageously, the use of an osmoprotectant as described herein, reduces the need for complex nutrients.

Specifically exemplified herein is the use of betaine as the osmoprotectant. Addition of betaine allows, for example, E. coli strains to produce increased yields of desired products as a result of the fermentation of 10% (w/v) sugar in mineral salts medium.

In a specific embodiment, addition of 1 mM betaine, a non-metabolized protective osmolyte, to Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol F ¹ h ^'1 ; 0.8 to 2.3 g I ^"1 h ^"1 ). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during D-lactate production. In mineral salts medium supplemented with betaine, SZl 32 produced approximately 1 mol D-lactate (90 g) per 100 g sugar (glucose or sucrose).

Improvement in acetate and pyruvate production are also exemplified herein.

The use of betaine, or other osmoprotectant as described herein, as a supplement for mineral salts media offers advantages over the use of complex additives including a reduction in cost associated with nutrients, product purification, and waste disposal.

BRIEF DESCRIPTION OF THE FIGURES

Figure IA- ID shows the effect of betaine (1 mM) on the growth-inhibition of W3110 by high concentrations of glucose and organic acids in mineral salts medium. A. Glucose; B. Pyruvate (with 2% w/v glucose, pH 7). C. Acetate (with 2% w/v glucose, pH 7); D. Acetate (with 2% w/v glucose, pH 8). Culture tubes (13x100 mm) containing 4 ml of mineral salts medium with glucose or with glucose (2% w/v) and salts of organic acids were inoculated to an initial density of 0.1 mg dry wt I ^'1 . After incubation at 37 ⁰ C for 24 h, cell mass (growth) was estimated by measuring optical density. Symbols for all: W3110 without betaine, o; W3110 with 1 mM betaine, •.

Figure 2A-2C shows the effect of betaine on the aerobic fermentation of glucose to acetate by TC36 in mineral salts medium containing 10% (w/v) glucose. Fermentations were maintained at pH 7 (37 ⁰ C, 350 rpm). A. Cell mass. B. Acetate. C. 2-Oxoglutarate. Symbols for all: no betaine, o ; 1 mM betaine, •.

Figure 3A-3C shows the effect of betaine on the aerobic fermentation of glucose to pyruvate by TC44 in mineral salts medium containing 10% (w/v) glucose. Fermentations were maintained at pH 7 (37 ⁰ C, 350 rpm). A. Cell mass. B. Pyruvate. C. 2-Oxoglutarate. Symbols for all: no betaine, o ; 1 mM betaine, •.

Figure 4A-4C shows the effect of betaine on growth and fermentation in mineral salts medium containing 10% (w/v) sugar (glucose or sucrose). Luria broth containing 10% (w/v) glucose was included for comparison in A and B. Fermentations were maintained at pH 7 (37 ⁰ C, 150 rpm). A. Production of organic acids from glucose. B. Cell mass during fermentation with glucose. C. Cell mass and organic acids during the sucrose fermentation. Symbols for A and B: no betaine, o ; 0.25 mM betaine, ▲ ; 0.50 mM betaine, * ^• ; 1 niM betaine, • ; 2 mM betaine, D ; and Luria broth, ■ . Symbols for C: cell mass without betaine, D ; lactate without betaine, o ; cell mass with 1 mM betaine, ■ ; lactate with 1 mM betaine, • .

Figure 5A-5B shows the effect of betaine (1 mM) on glucose and lactate tolerance in mineral salts medium. A. Glucose. B. Potassium lactate (with 2% w/v glucose). Culture tubes (13x100 mm) containing 4 ml of mineral salts medium with glucose or with glucose (2% w/v) and potassium lactate were inoculated to an initial density of 0.1 mg dry wt I ^"1 . After incubation at 37 ⁰ C for 24 h, cell mass (growth) was estimated by measuring optical density at 550 nm. Symbols for all: SZ132 without betaine, o; SZ132 with 1 mM betaine, •.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, osmoprotectants, such as betaine, may be used to increase the productivity and titer of anaerobic and aerobic fermentation with E. coϊi. and other microbes. Betaine serves as a protective osmolyte, during fermentations with high concentrations of sugars or products, replacing the need to divert carbon and energy into native osmoprotectants such as glutamate and trehalose. Thus, in accordance with the subject invention, betaine and/or other osmoprotectants can be added to media and improve the rates of biological conversion of sugars into chemicals such as organic acids. Osmoprotectants are well known to those skilled in the art and include such compounds as betaine, choline, dimethylsulfoniopropionate, mannitol, inositol, proline, fructan, trehalose, and D-ononitol.

The methods of the subject invention can be used, as discussed below for the efficient production or organic compounds such as, for example, lactate and pyruvate.

Lactate Production

In a specific embodiment, addition of 1 mM betaine, a non-metabolized protective osmolyte, to Escherichia coϊi B strain SZl 32 during batch fermentation in mineral salts

medium with 10% (w/v) sugar doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol I ^"1 h ^"1 ; 0.8 to 2.3 g I ^"1 h ^"1 ). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during D-lactate production. In mineral salts medium supplemented with betaine, SZl 32 produced approximately 1 mol D-lactate (90 g ) per 100 g sugar (glucose or sucrose).

Pyruvate production >

The addition of betaine to mineral salts medium dramatically improved the fermentation of 10% (w/v) glucose to pyruvate by strain TC44. With betaine, high titers of pyruvate (737 mM; 66 g I ^"1 ) were achieved in simple batch fermentations using mineral salts medium. Although higher titers have been reported with Torulopsis glabrata (68 g I ^'1 ; Liu et al. 2004, Liu et al. 2005), productivity was lower than with TC44 and required complex nutrients, acetate, oxaloacetate and vitamins. Using more complex fed batch processes, E. coli mutants have been shown to produce 79 g l ^"1 pyruvate (Zelic et al. 2004). With TC44 and betaine, pyruvate yields based on sugar metabolized (1.68 pyruvate per glucose), among the highest reported. Growth of TC44 in 10% (w/v) glucose was severely limited in the absence of betaine, presumably due to inability of this strain to produce sufficient levels of protective osmolytes such as trehalose and glutamate.

Strain TC44 did not grow well in mineral salts medium containing 10% (w/v) glucose in comparison to strains TC36 and W3110 although all three strains grew well in the same media containing a lower glucose concentration. Addition of 1 mM betaine caused a modest 2-fold increase in cell mass after 24 h for the latter strains and 10-fold increase in cell mass for strain TC44 (pyruvate production).

In mineral salts medium containing 10% glucose (556 mM), betaine addition promoted more rapid growth, higher cell yields, and higher glycolytic flux (and lactate production). Betaine had little effect on growth rate in 10% (w/v) sucrose (292 mM), presumably due to lower osmotic effect in comparison to glucose, but prolonged the growth phase to increase cell yield and volumetric productivity. Betaine reduced the detrimental effect of added lactate on growth and metabolism, allowing metabolism to continue long after the growth phase and increasing final titers to near 1 M in mineral salts media. Specific rates of lactate production in Luria broth were lower than in mineral salts medium supplemented with betaine during the fermentation of 10% (w/v) glucose.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1— PRODUCTION OF ACETATE AND PYRUVATE Materials and methods

Strains, media and growth conditions

Three strains of E. coli were used in this study. Strain W3110 (ATCC 27325) is a wild-type K- 12 strain and is the parent used to construct TC44(A(focA-pflB) Afrd δldhA AatpFH AsucA adhEv.FRT AackA ApoxB) and TC36(A(focA-pflB) Afrd AldhA AatpFH AsucA adhEv.FRT ). Strain TC36 was engineered for acetate production by introducing a series of mutations and deletions in W3110. Strain TC44 was constructed from TC36 by deleting the genes {AackA and ApoxB) essential for each of the two major acetate producing pathways. These strains do not contain any foreign genes and can be regarded as non GMO organisms. Cultures were grown at 37 ⁰ C in NBS mineral salts medium (pH 7) containing 10% glucose (Causey et al. 2004). Betaine (1 mM) was added as a supplement where indicated.

Effect of betaine on inhibition of growth by glucose, acetate, and pyruvate

Tube cultures were prepared with NBS mineral salts medium (2% glucose, with or without 1 mM betaine) and various concentrations of glucose, potassium pyruvate, or potassium acetate. After inoculation with W3110 (parent) and incubation for 24 h, growth was compared by measuring optical density at 550 nm (Purvis et al. 2005). In some experiments, medium was adjusted from pH 7 to pH 8 prior to inoculation.

Fermentations

Fermentations were carried out in 6.6-liter vessels with a 5-liter initial liquid volume (37 ⁰ C, dual Rushton impellers, 350 rpm) using New Brunswick Bioflow 3000 fermentors (Causey et al. 2004). Dissolved oxygen was maintained above 5 % of air saturation by

altering the proportion of O ₂ and air at a constant flow rate of 1.0 L min ^"1 (0.2 wm). Broth was maintained at pH 7 or pH 8 by automatic addition of 11.4 M KOH. Seed cultures were prepared by inoculating colonies from a fresh plate into NBS mineral salts medium containing 3% glucose and 0.1 M MOPS (Causey et al. 2004). Cells harvested from flask cultures were used to provide an inoculum of 16.5 mg dry cell weight I ^"1 .

Analyses

Cell mass was estimated from measurements of optical density at 550 nm (~1 g cdw I ^"1 at 3.0 OD). Total organic acid production (primarily acetate or pyruvate) was measured by base (KOH) usage to maintain pH. Acidic products were analyzed by HPLC (Zhou et al. 2003). Ethanol was measured by GC (Ohta et al.1991).

Average volumetric rates and maximum specific rates were estimated from measured values for glucose and acetate or pyruvate using GraphPad Prism (GraphPad Software, San Diego, CA). A smooth curve was generated with 10 points per min (Lowess method) to fit measured results. The first derivative (acetate or glucose versus time) of each curve served as an estimate of volumetric rate. Specific rates (mmol I ^"1 h ^"1 mg ^"1 dry cell weight) were calculated by dividing volumetric rates by respective values for cell mass.

Betaine increases the growth of WSIlO (parent) in media containing high concentrations of glucose, pyruvate and acetate

To achieve high product titers in simple batch fermentations, biocatalysts must function in mineral salts medium under osmotic stress from both the substrate and products. The effect of a protective osmolyte, betaine, on this stress was investigated in mineral salts medium (pH 7) using the parent strain, W3110. This strain was selected because TC36 and TC44 have mutations in oxidative phosphorylation and all major fermentation pathways, preventing their growth under anaerobic conditions (Causey et al. 2004). With W3110, addition of 1 mM betaine decreased the extent of growth-inhibition by high concentrations of glucose (Figure IA) and pyruvate (Figure IB) but did not alter inhibition by acetate (Figure 1C). Little growth was evident above 600 mM glucose without betaine. With betaine, growth was reduced but not prevented by 1 M glucose. The addition of betaine increased the MIC for pyruvate by 50%. The beneficial effect of betaine for growth at high glucose and pyruvate concentrations is consistent with osmotic stress as the primary factor limiting growth by these two compounds.

The low MIC for acetate (300 mM) in comparison to pyruvate (500 niM) suggest that acetate-inhibition involves other actions such as dissipation of δpH (Russell & DiezGonzalez). This hypothesis was tested by conducting experiments using medium adjusted to pH 8 prior to inoculation (Figure ID). Although the concentration of the neutral acid conjugate (the membrane-permeable species) is 10-fold lower at pH 8 than at pH 7, the MIC without betaine remained the same under both conditions. At pH 8, however, the addition of betaine doubled the MIC for acetate. These results indicate that the permeability of acetate is sufficiently low at pH 8 to cause osmotic stress. At pH 7, other detrimental actions of acetate appear to dominate (Roe et al. 2002, Russell & DiezGonzalez 1998). Beneficial effects of added betaine for glucose-tolerance and pyruvate-tolerance were similar at pH 7 and pH 8.

Production of pyruvate and acetate from 10% (w/v) glucose

Both strains TC44 and TC36 grew well and fermented 3-6% (w/v) glucose in minerals salts medium, producing pyruvate and acetate, respectively, as dominant products (Causey et al. 2003; Causey et al. 2004). However, only TC36 was able to effectively ferment 10% (w/v) glucose in the same mineral salts medium (Figures 2 and 3). With 10% (w/v) glucose, yields based on sugar metabolized were higher for TC44 but growth and fermentation were very slow (Table 1). On a molar basis, the final acetate titer with TC36 (685 mM; 41.1 g I ^"1 ) was 6-fold higher than the pyruvate titers with TC44 (114 mM; 10.6 g 1 ^" '). Since TC44 is an isogenic derivative of TC36, the poor performance of TC44 in 10% glucose can be presumed to result from loss of function mutations (ackA mdpoxB).

Table 1. Effect of betaine on metabolic rates ^a

Glycolytic Flux Acetate/Pyruvate

TC36 - 0.15 10 16 10 20

TC36 + 0.42 11 13 11 24

*TC36 - 0.16 7 12 11 14

*TC36 + 0.38 7 10 10 13

TC44 - 0.09 1 6 2 13

TC44 + 0.29 10 35 16 42

^a Conditions: NBS media containing 10% glucose (556mM),aerobic, 350 rpm, 37 ⁰ C, and betaine (ImM was added as indicated). Fermentations were controlled at pH 7 (unmarked) or pH8 (marked with an asterisk). ^b Average volumetric rates were calculated from 95% of the maximum concentration. ^c Maximum specific rates (calculated as described in materials and methods section).

Effect of betaine addition (1 mM) of the fermentation of 10% (w/v) glucose

Figure 1 shows that betaine reduced growth-inhibition by high concentrations of glucose and pyruvate at pH 7, and by acetate (pH 8 only). However, the addition of betaine was of limited value for the fermentation of 10% (w/v) glucose to acetate by strain TC36 at either pH (Tables 1 and 2; Figure 2). Betaine increased the initial rates of growth and fermentation but also increased unwanted co-products (Figure 2; Table 2). Co-product accumulation (2-oxoglutarate and succinate) began at the end of the growth phase (Figure 2C), decreasing acetate yield (Table 2). Betaine had little effect on volumetric and specific rates of acetate production or glycolytic flux (Table 1).

Table 2. Effect of betaine on fermentation products and yield ^a

Cell Yield ^b Organic Acids (mmoi r ¹ ) ⁰

Strain Betaine Mass Pyruv. or Acet.

(I mM) (g r ¹ ) (% theo. yield)

Pyruv. Acet. 2-Oxoglut. Sue.

TC36 - 3.03 57 207 685 28 2

TC36 + 4.02 49 157 552 86 4

*TC3 - 2.98 75 27 727 1 <1 6

*TC3 + 3.85 77 <1 675 66 14 6

TC44 0.46 55 114 <1 <1

TC44 + 3.40 78 761 23 81 ^a Conditions: NBS media containing 10% glucose (556mM), aerobic, 350rpm, 37 ⁰ C. Betaine (1 mM) was added as indicated. Fermentations were at pH 7 (unmarked) or pH 8 (marked with an asterisk).

''The maximum theoretical yield for pyruvate is 0.978 g pyruvate (g glucose) ^" \ The maximum theoretical yield for acetate is 0.67g acetate (g glucose) ^'1 .

⁰ Abbreviations: Pyruv., pyruvate; Acet., acetate; 2-Oxoglut, 2-oxoglutarate; Sue, succinate.

In contrast to TC36, the addition of 1 mM betaine dramatically improved growth and pyruvate production by TC44 in mineral salts medium containing 10% (w/v) glucose (Figure 3; Tables 1 and 2). Betaine addition increased the maximum growth rate by 3 -fold, glycolytic flux by 6-fold, specific pyruvate production by 3.5 -fold, and volumetric productivity by 8- fold (Table 1). With added betaine, TC44 completed the fermentation of 10% (w/v) glucose in 60 h, a shorter time than required by TC36 with or without betaine. The maximum growth rate and productivities for TC44 with added betaine were higher in all cases than those for TC36 (Table 1).

EXAMPLE 2— PRODUCTION OF D-LACTATE Materials and methods

Strains, media and growth conditions

Strain SZ132 (A(focA-Z. mobilis pdc-adhB-pflB) adhEv.FRT δackA::FRT tψdr.K. oxytoca casAB lacYr.E. chrysanthemi celY ) (Zhou et al. 2005) was previously constructed from E. coli KOI l, an ethanologenic derivative of E. coli B (ATCC 11303). Strain SZ132 produces D-lactate as the primary fermentation product from hexose and pentose sugars, and has the native ability to ferment sucrose. Cultures were grown at 37 ⁰ C in NBS mineral salts medium (Causey et al. 2004) or Luria broth (per liter: 1O g tryptone, 5 g yeast extract, and 5 g NaCl) supplemented with 2%-10% (w/v) glucose or sucrose.

Inhibition of growth by glucose and lactate

Growth was compared in culture tubes with NBS mineral salts medium containing the indicated concentrations of glucose, lactate, and betaine, essentially as described previously (Purvis et al. 2005). Cell mass was estimated by measuring optical density at 550 nm after incubation for 24 h (37 ⁰ C).

Fermentations

Seed cultures were prepared as described previously (Shukla et al. 2004; Zhou et al. 2003; Zhou et al. 2005) and used to inoculate small fermentation vessels (350 ml working volume, 37 ⁰ C, 150 rpm agitation, inoculum dry wt of 33 mg I ^"1 ). Broth was maintained at pH 7.0 by the automatic addition of 6 M KOH. Plotted data represent an average of 2 or more replicates. Bars denoting the SεM are included for averages of 3 or more experiments. Specific growth rate was estimated from the steepest region of the curve, the initial 6 h period in most cases. D-lactate volumetric productivity (maximum) was calculated for the initial 12 h period of fermentation in most cases. Specific productivity was calculated by dividing this rate by the average cell mass. Since D-lactate is the primary fermentation product, specific rates of lactate production are representative of glycolytic flux. Analyses

Cell mass was estimated from measurements of optical density at 550 nm. Acid production (primarily lactate) was measured by base (KOH) usage to maintain pH 7.0. Acidic

products were analyzed at the end of fermentation by HPLC (Zhou et al. 2003). Ethanol was measured by GC (Ohta et al.1991).

Results

Betaine decreased the time required to ferment of 10% (w/v) glucose by SZl 32 in mineral salts medium

Supplementing mineral 'salts medium containing 10% (w/v) glucose with betaine resulted in a 60% increase in the maximum specific growth rate (Figure 4; Table 3), and dose-dependent increases in cell yield due to an extension of the growth phase. Betaine addition also increased specific and volumetric lactate production (Figure 4; Table 3).

Table 3. Effect of betaine on growth rate and lactic acid productivity by SZl 32

Media, sugar Betaine Max. spec. ^a Max vol. prod. ^b Max. spec, prod. ⁰ Max. vol. prod. ^b

(mM) • growth rate (mmol I ^'1 ) K ¹ (mmol g ) K ¹ (g r') h- ¹ μ (D/h)

LB, glucose 0 0.46 51.0 27.2 4.6

NBS, glucose 0 0.14 8.6 26.6 0.8

NBS, glucose 0.25 0.23 19.6 36.0 1.8

NBS, glucose 0.50 0.23 22.1 37.6 2.0

NBS, glucose 1.00 0.23 24.5 36.0 2.2

NBS, glucose 2.00 0.23 25.7 40.2 2.3

NBS, sucrose 0 0.07 8.0 23.5 0.7

NBS, sucrose 1.00 0.08 14.6 16.3 1.3

^a Maximum specific growth rate. ^b Maximum volumetric productivity per liter. ^c Maximum specific productivity

Since lactate is the dominant product of fermentation representing approximately 90% of glucose carbon with SZ 132 (Table 4), increased specific lactate production (Table 3) can be regarded as an increase in glycolytic flux.

Table 4. Fermentation of glucose and sucrose by SZ132

Lactate Co-products ^d

Media, sugar ^a Residual Time ^b

Yield Yield ^c Succinate Acetate sugar (h) (ήimol per (niM) (%) (rnM) (niM) 100 g sugar)

LB, glucose 0 48 1000 ± 18 90 35.2 ± 1.9 12.5 ± 1.0

NBS, glucose 210 >168 614 ± 1 55 32.1 ± 1.5 14.0 ± 0.6

NBS, glucose 6 72 930 ± 9 84 78.9 ± 2.4 11.6 ± 0.8 + 1 HiM betaine

NBS, sucrose 131 >168 610 ± 33 52 2.8 ± 0.0 < 1

NBS, sucrose 0 96 1046 ± 158 89 38.0 ± 7.3 12.2 ± 3.7 + ImM betaine

Conditions: pH 7, 37 ⁰ C, 150 rpm, 10% (w/v) sugar (556 rnM glucose; 292 rnM sucrose) and either NBS mineral salts or LB (Luria broth, a complex medium). ^b Estimated incubation time required for greater than 90% sugar utilization. ^c Percentage of maximum lactate yield: 2 mol per mol hexose (glucose or fructose) and 4 mol per mol sucrose. Reported yields were calculated based on total sugar present. ^d Ethanol was less than 1 mM in all fermentation broths.

Specific rates of lactate production (and glycolytic flux) were approximately equal for mineral salts medium containing 10% (w/v) glucose without betaine and Luria broth. With supplemental betaine, specific rates of lactate production (and glycolytic flux) were up to 50% higher for mineral salts medium than for Luria broth. However, volumetric productivity for Luria broth cultures was higher than for betaine-supplemented mineral salts due to more rapid cell growth and higher cell yields in rich medium.

The addition of 1 mM betaine to mineral salts medium with 10% (w/v) glucose doubled the level of succinate, reducing D-lactate yield (Table 4). Without betaine, co- product levels in Luria broth and mineral salts medium were similar. No ethanol was detected in any of these fermentations by HPLC or GC. However, the chiral purity of D-lactate was reduced from 99.5% to 95% by betaine addition.

Betaine reduced the inhibition of growth by glucose and lactate in mineral salts medium

Betaine improved growth and fermentation performance of SZl 32 in mineral salts medium containing 10% (w/v) glucose. To determine the basis of this effect, growth inhibition by glucose and lactate were compared in the presence and absence of betaine. Supplementing with 1 mM betaine was beneficial for glucose and lactate tolerance (Figure 5). Betaine increased the growth of SZl 32 in all glucose concentrations above 400 mM, and increased the MIC for glucose from approximately 800 mM to 1000 mM. With 10% (w/v) glucose (556 mM), cell mass after 24 h was only 30% of that with 2% (w/v) glucose. Smaller but significant benefits were observed for betaine during growth in the presence of 100-400 mM potassium lactate (Figure 5B). Betaine addition caused a small increase in the MIC for lactate.

Betaine (1 mM) increased the rate and extent of sucrose fermentation in mineral salts medium

Strain SZ132 grew and fermented sucrose more slowly than glucose in mineral salts medium containing or lacking betaine (Figure 4C). Betaine had little effect on initial growth rate with 10% (w/v) sucrose (292 mM), but prolonged the growth phase resulting in a higher cell yield and higher volumetric productivity. Maximum specific productivities with and without betaine were similar, consistent with the lower osmotic stress associated with sucrose. With 1 mM betaine, 10% (w/v) sucrose was fermented to completion in 120 h. Without betaine, this level of sucrose was not metabolized to completion even after 1 week of incubation. Over 1 mole of lactate was produced per 100 g of sucrose.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.