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Engineering Lactic Acid Bacteria with Pyruvate Decarboxylase and Alcohol Dehydrogenase Genes for Ethanol Production from Zymomonas Mobilis

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Engineering Lactic Acid Bacteria with Pyruvate Decarboxylase and Alcohol Dehydrogenase Genes for Ethanol Production from Zymomonas Mobilis J Ind lvlicrobiol Biotechnol (2003) 30: 315-321 860 DOl 10.1007s10295-003-0055-z ORIGINAL PAPER Nancy N. Nichols' Bruce S. Dien . Rodney J. Bothast Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis Received: 12 December 2002 Accepted: 12 March 2003 Published online: 15 May 2003 © Society for Industrial l"licrobiology 2003 Abstract Lactic acid bacteria are candidates for eng:i­ Introduction neered production of ethanol from biomass because th~y are food-grade microorganisms that can. in many case;. Agricultural biomass has the potential to supplement metabolize a variety of sugars and grow unde;' harsh starch as a feedstock for fuel ethanol production. conditions. In an effort to divert fermentation from Hydrolysis of the lignocellulose in biomass yields a production of lactic acid to ethanoL plasmids were mixture of hexose and pentose sugars. including glucose. constructed to express pyruvate decarboxylase (PDC) galactose, xylose. and arabinose. However, the current and alcohol dehydrogenase (ADH), encoded by the pdc starch-based technology has not yet been extended to a and adhB genes of Zymomonas mobilis, in lactic acid biomass-to-ethanol process. in part because the veast bacteria. Several strains were transformed with the Saccharomyces cerevisiae does not metabolize pentoses. plasmids. and transcription of pdc and adhB was con­ Naturally occurring yeasts that ferment xylose have lmv firmed by northern hybridization analvsis of transfor­ ethanol yields and production rates [3]. ·Consequently. mants. PDC and ADH enzyme activities were at least ethanol fermentation from biomass-derived sugars must 5- to 10-fold lower in these bacteria compared to employ new fermentative microorg:anisms with an Escherichia coli transformed with the same plasmid. expanded substrate range. ~ Glucose fermentations were carried out, and some. but Recombinant organisms have been constructed that not alL of the transformed strains produced more eth­ ferment sugar mixtures selectivelv to ethanol. These in­ anol than the untransformed parent strains. However, clude S. cerevisiae [20] and the Gram-negative bacteria lactic acid was the primary fermentation product formed Zymomonas mobilis [31]. Escherichia coli [10. 21]. and by all of the transformants, indicating: that ADH and Klebsiella oxytoca [24]. Gram-positive bacteria are an­ PDC activities were insufficient to divert sig:nificant other group with the potential for ethanol production. carbon flow towards ethanoL ~ Many of these microorganisms have the abilitv to metabolize a variety of sugars. including pentose su~ars. Keywords Ethanol' Pyruvate decarboxylase' Alcohol Gram-positive bacteria are widelv used in the food dehydrogenase' Lactic acid bacteria' Molecular industry. and thus are generally rec~gnized as safe. Some biology grow at low pH and at 50°C or higher; these traits \vould allow adaptation to current fermentation systems. which would otherwise be vulnerable to contamination. In addition. many strains are relatively tolerant to ethanol. Some lactobacilli have been identified that are capable of N. N. Nichols (I>.q . B. S. Dien . R. J. Bothast Fermentation Biotechnologv Research Unit \:ithstanding 16% (v/v) ethanol [16]. Thus. Gram-posi­ National Center for Agric~itura1 Utilizatiol; Research. tIve ethanologens would offer a unique combination of Agricultural Research Service. USDA. desirable traits. 1815 North University Street. Peoria. IL 61604. USA This study focused on the lactic acid group of Gram­ E-mail: nicho1nn(i[ ncaur.usda. gOV positive bacteria. Homofermentative lactic acid bacteria Tel.: 1-309-6816271 - Fax: + 1-309-6816427 ferment glucose to lactate via reduction of pyruvate by lactate dehydrogenase (LDH). Heterofennentative Preselll address: R. J. Bothast strains also produce acetate. ethanol. and CO via phos­ National Corn-to-Ethano1 Research Pilot Plant. 2 Southern Illinois University-Edwardsville phoketolase pathway activity. In engineered strains. Edwardsville. IL 62025. USA . expression of pyruvate decarboxylase (PDq and alcohol 316 dehvdrogenase (ADH) could divert carbon flow to etha­ ration [4]. Transformation was verified by PCR screening of nol"rath;r than to lactate. The central metabolite pyru­ erythromycin-resistant colonies, using oligonucleotide primers from the Z. lIlobilis pde gene sequence [6] to detect the presence of vate is converted to acetaldehyde by PDC and the recombinant plasmids. Plasmid DNA was prepared with the subsequently to ethanol by ADH. This strategy has been Qiaprep Spin Kit (Qiagen. Valencia. Calif.). For Gram-positive applied successfully to E. coli [10, 21] using the pet bacteria. cells from 10-ml overnight cultures were incubated with operon, which carries the pdc and adhB genes from 2 mg ml I Ivsozvme for 5 min at 37°C. and plasmid DNA was isoh~ted acc;rdi~g Z. mobilis. The pet operon has also been placed in the to the manufacturer's protocol. Cloned DNA was sequenced with the Big Dye Terminator Ready Reaction Kit Gram-positive bacteria Bacillus [2] and Lactobacillus (Applied Biosystems, Foster City, Calif.). casei [17], but selective production ofethanol has not been Two plasmids (pNETI25 and pNETI31: Fig. I) were con­ achieved. In work described here. two variations of the structed to express PDC and ADH in lactic acid bacteria. First. the pet operon were constructed that have expression signals fragment of DNA containing the pde and adhB genes was cloned by PCR amplification from pLOI297 [I] so that the pde start codon is appropriate for lactic acid bacteria, and several strains present within an engineered Ndd site. on a construct named were transformed with each plasmid. Gene expression, pNETIOI. The cloned PCR product contained the pde and adhB PDC and ADH enzyme activities, and ethanol yields \vere genes. beginning with the pde start codon and ending four bases compared for the transformed and parent strains. downstream of the adhB stop codon: this included the intergenic region that was created when the unlinked pde and adhB genes were combined to form the pel operon [21]. The primers used for amplification were 5'-GCATCATATGAGTTATACTGTCGG­ Materials and methods TACC-3' (spanning the 5' end of pde: start codon underlined) and 5'-GAAGCCCGGGAAATTAGAAAGCGCTCAGG-3' (spanning the 3' end of adhB: stop codon on opposite strand underlined). Bacterial strains. culture media. and growth conditions Two promoters (Fig. I) were cloned separately, then joined to the Z. lIlobilis pde start codon at the Ndd site. One promoter se­ Bacterial strains used in this studv are described in Table I. E. coli quence (in pNETI25: Fig. IA) was PCR-amplified from the was grown at 37°C aerobicallv in"BHI medium (Becton Dickinson, Slreploeoeells boris Idh gene on pUCLDH I [30] using primers Sparks. Md.) and anaerobiG~lly as described by Dien et al. [10]. 5'-ACTAGTTTATTCGTGACATTTCTCACTTAACG-3' and 5'­ Where appropriate. media were supplemented with ampicillin. CATATGTTTCCTCCTTGGTTTACAAGTGCG-3' (NdeI recog­ chloramphenicol. erythromycin. or kanamycin at 100.20. 150, and nition sequence underlined). The amplified region encompassed the 50 mg I-I. respectivelv. Lactic acid bacteria were grown in MRS 5' end of the Idh gene in order to couple translation of the pel genes medi~m (Becton Dickinson) supplemented with 10 mg I-I eryth­ to a fragment of the Idh open reading frame. A consensus ribosome­ romvcin for transformed strains. LaelOeoeelis laC/is was grown at binding site (RBS) was added [8]. and the Idh gene was truncated 30°C. Other strains were grown at 37°C. Cells used 1'0; enzvme after 30 residues bv the addition of a stop codon. designed to assavs were grown anaer;bicallv for 18-24 h in 100 ml rV[RS overlap the pde start codon. The modified S. boris promoter region flush~d ga~ medium with nitrogen and containing 500 mg I-I cys­ was fused to the pde start codon. forming the final expression teine HCI. in glass bottles sealed with butyl rubber stoppers. Fer­ construct pNET125. This plasmid has the S. boris promoter plus the mentation experiments were conducted in triplicate in 125 ml pel genes. in the low copy shuttle vector pTRKL2 [25]. Erlenmever flasks. unless stated otherwise. MRS medium (50 ml) The second promoter sequence (in pNET13I: Fig. IB) has a was supplemented with 40 g I-I glucose (resulting in 60 g I-I total consensus L. laelis promoter (CP40). with optimized sequences glucose. because l'v[RS contains 20 g I-I), and buffered with 9 g flanking the consensus - 35 and -10 regions [23]. and an added RBS CaC03 (marble chips 6210, Mallinckrodt. SI. Louis, Mo.) per flask. sequence. The promoter sequence represented in Fig. IB was con­ The flasks were capped with rubber stoppers vented with 22 gauge structed from complementary single-stranded oligonucleotides needles. Cultures were gently agitated to promote mixing of the (20 llM each). which were annealed in 25 m]\'[ Tris, pH 8 and carbonate. and sampled after 72 h. LaelObaeillus plalllarlllll TFI03 I0 I~1M lv[gCk and cooled from 95°C to room temperature over (an Idh mutant that grows best under anaerobic culture conditions 2 h. The al;ne,~led double-stranded fragment was used for blunt-end [12]) and its transfo'i-mants were grown for fermentation experi­ ligation without further purification. The cloned promoter region ments anaerobically as described above. was fused to the pel genes at the introduced Ndd site (Fig. IB). and the entire region spanning the promoter plus pel genes was cloned into the high-copy shuttle vector pTRKH2 [25]. to form pNETI31. Plasmid construction PCR. li!wtions. and E. coli transformations were performed by RNA analyses standard techniques [27]. PCR products were cloned using the TA Cloning Kit (Invitrogen, Carlsbad. Calif.). Competent strains of Bacterial RNA was isolated from activelv growing cultures. Cell lactic acid bacteria were prepared and transformed by electropo- pellets were suspended in buffer containing to m1'v[ ~\10PS. pH 7.3 Table I Bacterial strains Strain Description Source or Reference Escherichia coli INV:zF' Cloning strain Invitrogen (Carlsbad.
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