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Abstract: Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low‐pH production of d‐lactic acid (LA), an acid‐tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d‐lactate dehydrogenase gene (d‐LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole‐genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d‐LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively. [ABSTRACT FROM AUTHOR]

ABSTRACT Xylose fermentation by engineered Saccharomyces cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD+-linked xylitol dehydrogenase (XDH) suffers from redox imbalance due to cofactor difference between XR and XDH, especially under anaerobic conditions. We have demonstrated that coupling of an NADH-dependent acetate reduction pathway with surplus NADH producing xylose metabolism enabled not only efficient xylose fermentation, but also in situ detoxification of acetate in cellulosic hydrolysate through simultaneous co-utilization of xylose and acetate. In this study, we report the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast with XR and XDH through optimization of the acetate reduction pathway. Specifically, we constructed engineered yeast strains exhibiting various levels of the acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) activities. Engineered strains exhibiting higher activities of AADH and ACS consumed more acetate and produced more ethanol from a mixture of 20 g/L of glucose, 80 g/L of xylose, and 8 g/L of acetate. In addition, we performed environmental and genetic perturbations to further improve the acetate consumption. Glucose-pulse feeding to continuously provide ATPs under anaerobic conditions did not affect acetate consumption. Promoter truncation of GPD1 and gene deletion of GPD2 coding for glycerol-3-phosphate dehydrogenase to produce surplus NADH also did not lead to improved acetate consumption. When a cellulosic hydrolysate was used, the optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol with consumption of 4.1 g/L of acetate than a control strain without the acetate reduction pathway. These results suggest that the major limiting factor for enhanced acetate reduction during the xylose fermentation might be the low activities of AADH and ACS, and that the redox imbalance problem of XR/XDH pathway can be exploited for in situ detoxification of acetic acid in cellulosic hydrolysate and increasing ethanol productivity and yield. Biotechnol. Bioeng. 2016;113: 2587-2596. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Rapid advances in the capabilities of reading and writing DNA along with increasing understanding of microbial metabolism at the systems-level have paved an incredible path for metabolic engineering. Despite these advances, post-translational tools facilitating functional expression of heterologous enzymes in model hosts have not been developed well. Some bacterial enzymes, such as Escherichia coli xylose isomerase (XI) and arabinose isomerase (AI) which are essential for utilizing cellulosic sugars, cannot be functionally expressed in Saccharomyces cerevisiae. We hypothesized and demonstrated that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells might be the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co-expression of E. coli GroE can facilitate the functional expression of E. coli XI and AI, as well as the Agrobacterium tumefaciens D-psicose epimerase in S. cerevisiae. The co-expression of bacterial chaperonins in S. cerevisiae is a promising post-translational strategy for the functional expression of bacterial enzymes in yeast. Biotechnol. Bioeng. 2016;113: 2149-2155. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Reed ( Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium-mediated transformation using mature seed-derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector p IG121 Hm, were used to optimize T-DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T-DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μ m acetosyringone containing medium. Putative transformants were selected in 25 mg l−1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT-PCR and GUS staining of stems and leaves. [ABSTRACT FROM AUTHOR]

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein- O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene ( HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal. The GenBank Accession No. for the H. polymorpha PMT1 sequence is AY701415. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. The nucleotide sequence of the P. ciferrii SUR2 gene was submitted to GenBank under Accession No. AY151276. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]