Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Biofilm and metallo beta-lactamase production among the strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> spp. at a Tertiary Care Hospital in Kathmandu,Nepal

Introduction

Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL) among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients.

Methods

A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production.

Results

Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05%) were biofilm producers according to tube adherence test while, only 13 (15.29%) were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14%) isolates were biofilm producers on the basis of tube adherence test, while only 5 (10%) were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin.

Conclusion

In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.

     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

Equisetin as potential quorum sensing inhibitor of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Objective

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.

Results

QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.

Conclusions

Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

     

An NB-LRR gene, <Emphasis Type="Italic">TYNBS1</Emphasis>, is responsible for resistance mediated by the <Emphasis Type="Italic">Ty</Emphasis>-<Emphasis Type="Italic">2 Begomovirus</Emphasis> resistance locus of tomato

Key message

An NB-LRR gene, TYNBS1, was isolated from Begomovirus-resistance locus Ty-2. Transgenic plant analysis revealed that TYNBS1 is a functional resistance gene. TYNBS1 is considered to be synonymous with Ty-2.

Abstract

Tomato yellow leaf curl disease caused by Tomato yellow leaf curl virus (TYLCV) is a serious threat to tomato (Solanum lycopersicum L.) production worldwide. A Begomovirus resistance gene, Ty-2, was introduced into cultivated tomato from Solanum habrochaites by interspecific crossing. To identify the Ty-2 gene, we performed genetic analysis. Identification of recombinant line 3701 confirmed the occurrence of a chromosome inversion in the Ty-2 region of the resistant haplotype. Genetic analysis revealed that the Ty-2 gene is linked to an introgression encompassing two markers, SL11_25_54277 and repeat A (approximately 200 kb). Genomic sequences of the upper and lower border of the inversion section of susceptible and resistant haplotypes were determined. Two nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR) genes, TYNBS1 and TYNBS2, were identified around the upper and lower ends of the inversion section, respectively. TYNBS1 strictly co-segregated with TYLCV resistance, whereas TYNBS2 did not. Genetic introduction of genomic fragments containing the TYNBS1 gene into susceptible tomato plants conferred TYLCV resistance. These results demonstrate that TYNBS1 is a functional resistance gene for TYLCV, and is synonymous with the Ty-2 gene.

     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Species of family <Emphasis Type="Italic">Promicromonosporaceae</Emphasis> and family <Emphasis Type="Italic">Cellulomonadeceae</Emphasis> that produce cellulosome-like multiprotein complexes

Objectives

To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.

Results

Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.

Conclusion

This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.

     

A mutant in the <Emphasis Type="Italic">CsDET2</Emphasis> gene leads to a systemic brassinosteriod deficiency and <Emphasis Type="Italic">super compact </Emphasis>phenotype in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Key message

A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase.

Abstract

Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

Characterization of rhamnolipid biosurfactants produced by recombinant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain DAB with removal of crude oil

Objectives

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.

Results

Strain DAB had a higher yield of 17.3 g rhamnolipids l^?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l^?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m^?1 with CMC of 90 mg l^?1. The predominant rhamnolipid congeners were Rha–C₁₀–C₁₀ and Rha–Rha–C₁₀–C₁₀ detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.

Conclusion

Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

Drastic anthocyanin increase in response to <Emphasis Type="Italic">PAP1</Emphasis> overexpression in <Emphasis Type="Italic">fls1</Emphasis> knockout mutant confers enhanced osmotic stress tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress.

Abstract

Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Improvement of 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> by moderate expression of <Emphasis Type="Italic">puuC</Emphasis> (encoding an aldehyde dehydrogenase)

Objective

To improve 1,3-propanediol production in Klebsiella pneumoniae, the effects of puuC expression in lactate- and lactate/2,3-butanediol-deficient strains were assessed.

Results

Overexpression of puuC (encoding an aldehyde dehydrogenase) inhibited 1,3-propanediol production and increased 3-hydroxypropionic acid formation in both lactate- and lactate/2,3-butanediol-deficient strains. An improvement in 1,3-propanediol production was only achieved in a lactate-deficient strain via moderate expression of puuC; at the end of the fermentation, 1,3-propanediol productivity increased by 14 % compared with the control. Further comparative analysis of the metabolic flux distributions in different strains indicated that 3-hydroxypropionic acid formation could play a considerable role in cell metabolism in K. pneumoniae.

Conclusion

An improvement in 3-hydroxypropionic acid formation would be beneficial for cell metabolism, which can be accomplished by enhancing 1,3-propanediol productivity in a lactate-deficient strain via moderate expression of puuC.

     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.