Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces lactis

The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short- and long-term culture. The vectors carried an expression/secretion cassette consisting of the Saccharomyces cerevisiaeSUC2 gene under the control of the S. cerevisiaeα-factor promoter and leader. The first set of vectors contained the entire pKD1 sequence linearized at either the unique EcoRI or the unique SphI site of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity rapidly dropped while the plasmid-bearing population increased from 60% to 100%. This apparently contradictory behavior was due to structural instability. The enzyme restriction patterns of recovered plasmid DNA retained the pKD1 band while the band containing the SUC2 cassette had decreased substantially in size. To overcome this structural instability, a vector carrying the pKD1 replication origin and the cis-acting stability locus (lacking the inverted repeats) was employed in a pKD1⁺ (but otherwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While the new vector was significantly more stable, initial invertase activity was substantially lower than that for the vectors containing the full pKD1 sequence. Southern hybridization confirmed that this decrease was primarily due to reduced copy number. The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suitable for long-term (e.g. fed-batch or continuous) culture. Received: 24 June 1997 / Received revision: 14 November 1997 / Accepted: 29 November 1997     

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori⁺) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori⁺ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10⁻² copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest. Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed. (c) 1993 John Wiley & Sons, Inc.     

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid. Received: 27 March 1997 / Received revision: 13 August 1997 / Accepted: 25 August 1997     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis

A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal θ-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 °C and 37 °C, whereas derivatives that lack cisE were highly unstable at 37 °C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal θ-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 °C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 °C. Received: 30 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

3-Ketoglycoside-mediated metabolism of sucrose in E. coli as conferred by genes from Agrobacterium tumefaciens

Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc⁺ E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli. Received: 13 September 1996 / Received revision: 15 January 1997 / Accepted: 24 January 1997     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Selection of a subtilisin-hyperproducing Bacillus in a highly structured environment

Mutants that secrete increased amounts of enzyme into a selection medium can be efficiently enriched from large populations of mutagenized microorganisms during growth in hollow fibers. Under these conditions, each colony grows in its own microenvironment and cross-feeding between neighboring colonies is limited. We applied the technique to B. subtilis carrying a plasmid-encoded protease gene. The plasmid was subjected to random mutagenesis and clones secreting up to fivefold-increased amounts of enzyme were selected using a medium containing bovine serum albumin as the sole nitrogen source. Received: 22 May 1997 / Received revision: 21 October 1997 / Accepted: 7 November 1997     

Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured. (c) 1993 Wiley & Sons, Inc.     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Construction of a low-serine-type-carboxypeptidase-producing mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion

Using an antisense control strategy, we isolated an Aspergillus oryzae mutant that produced low levels of carboxypeptidases (CPases). The mutant TF_C-1 expressed the antisense RNA of the structural gene of CPase O and showed about 30% of the CPase activity in the parent strain. Gel filtration analysis indicated that this mutant decreased the CPase activities not only of CPase O but also of CPase O-1 and O-2. This result indicated that the antisense RNA was able to control the expression of the CPase genes as a group. Using the mutant as a heterologous protein expression host that produced the low levels of CPases, a stable and higher level of lysozyme expression could be obtained compared with the wild-type. In vitro proteolytic degradation assay also demonstrated that human lysozyme was degraded by purified CPase O. Received: 16 June 1997 / Received last revision: 29 August 1997 / Accepted: 15 September 1997     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme

By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-l bioreactor at 37 °C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 °C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26 000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammo-nium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme. Received: 18 February 1997 / Received revision: 27 March 1997 / Accepted: 27 March 1997     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997     

High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium

The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins. Received: 22 July 1998 / Received revision: 25 November 1998 / Accepted: 29 November 1998